Abrogen polypeptides, nucleic acids encoding them and methods for using them to inhibit angiogenesis

ABSTRACT

The present invention relates to novel nucleic acids encoding novel amino acid fragments of polypeptides, called abrogens. The present invention also relates to novel, potent in vitro and in vivo inhibitors of endothelial cell proliferation, and compositions of them and their use. The present invention further provides methods for modulating angiogenesis and/or inhibiting unwanted angiogenesis. Polypeptides according to the present invention are useful for developing cell growth-modulating compositions and methods and for treating and/or preventing cancer, tumor growth, or other angiogenic dependent or angiogenesis associated diseases.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of and claims priority to U.S. application Ser. No. 10/233,675, filed Sep. 4, 2002, and claims priority to U.S. provisional application No. 60/316,300, filed Sep. 4, 2001. The entire contents of each of the prior applications are specifically incorporated herein by reference.

FIELD OF THE INVENTION AND INTRODUCTION

[0002] The present invention relates to novel nucleic acids encoding novel amino acid fragments of polypeptides, called abrogens. The present invention also relates to novel, potent in vitro and in vivo inhibitors of endothelial cell proliferation, and compositions of them and uses of them. The present invention further provides methods effective for modulating angiogenesis and/or inhibiting unwanted angiogenesis. Polypeptides according to the present invention are useful for treating and/or preventing cancer, tumor growth, or other angiogenic dependent or angiogenesis associated diseases.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis is the generation of new blood vessels from preexisting vessels into a tissue or organ. Angiogenesis is required and observed under normal physiological conditions, such as in wound healing, fetal and embryonic development, female reproduction, i.e., formation of the corpus luteum, endometrium and placenta, organ formation, and tissue regeneration and remodeling (Risau W et al., Nature, 1997, 386, 671-674).

[0004] Angiogenesis begins with local degradation of the basement membrane of capillaries followed by invasion of stroma by underlying endothelial cells in the direction of an angiogenic stimulus. Subsequent to migration, endothelial cells proliferate at the leading edge of a migrating column and then organize to form new capillary tubes.

[0005] Persistent, unregulated angiogenesis occurs in a multiplicity of pathological conditions, tumor metastasis and abnormal growth by endothelial cells, and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases. Outgrowth of new blood vessels under pathological conditions can lead to the development and progression of diseases such as tumor growth, diabetic retinopathy, tissue and organ malformation, obesity, macular degeneration, rheumatoid arthritis, psoriasis, and cardiovascular disorders.

[0006] Several studies have produced direct and indirect evidence that tumor growth and metastasis are angiogenesis-dependent (Brooks et al., Cell, 1994, 79, 1154-1164; Kim KJ et al., Nature, 1993, 362, 841-844). Expansion of the tumor volume requires the induction of new capillary blood vessels. Tumor cells promote angiogenesis by the secretion of angiogenic factors, in particular basic fibroblast growth factor (bFGF) (Kandel J. et al., Cell, 1991, 66, 1095-1104) and vascular endothelial growth factor (VEGF) (Ferrara et al., Endocr. Rev., 1997, 18: 4-25). Tumors may produce one or more of these angiogenic peptides that can synergistically stimulate tumor angiogenesis (Mustonen et al., J Cell Biol., 1995, 129, 865-898). Therefore, expression or administration of anti-angiogenic factors, by gene therapy, for instance, should counteract the tumor-induced angiogenesis.

[0007] Various anti-angiogenic polypeptides have been discussed and used to treat human angiogenic dependent or angiogenic associated diseases. For example, angiostatin and endostatin are proteolytic fragments of plasminogen (Pgn) and collagen XVIII, respectively (O'Reilly et al., Cell, 1994, 79:315-328; O'Reilly et al., Cell, 1997, 88:1-20). Angiostatin contains the first four disulfide-linked structures of plasminogen, which are known as kringle structures, and which display differential effects on the suppression of the endothelial cell growth. For example, kringle 1 was shown to exhibit some inhibitory activity, while kringle 4 is an ineffective fragment. Hua L et al., (BBRC, 1999, 258 :668-673) has characterized another kringle structure within plasminogen but outside of angiostatin, e.g., kringle 5. The kringle 5 was shown to inhibit endothelial cell proliferation and migration. Also, Renhai et al. (PNAS, 1999, Vol. 96, No. 10, pp. 5728-5733) has demonstrated a synergistic effect on endothelial inhibition when angiostatin and kringle 5 were coincubated with capillary endothelial cells. It was, however, stated that such association did not completely arrest tumor growth or tumors at a dormant stage.

[0008] The prothrombin kringle-2 domain, which is a fragment released from prothrombin by factor Xa cleavage, was identified as having anti-endothelial cell proliferative activity by Lee TH et al. (JBC, 1998, vol 273, No. 44, pp. 25505-25512) using in vitro angiogenesis assay system with bovine capillary endothelial (BCE) cell proliferation. The prothrombin kringle-2 domain was, however, described as having endothelial cell suppression activities comparable with those of angiostatin.

[0009] An amino terminal portion of the urokinase plasminogen activator uPA, termed ATF, has also been disclosed (Li et al., Hum Gen Ther 10: 3045-53, 1999; Griscelli et al., Hum. Gen. Ther., 1999, Vol 10, No. 18, pp. 3045-53) as inhibiting angiogenesis. uPA is composed of three domains, a serine proteinase domain, a kringle domain, and a growth-factor-like domain. The urokinase plasminogen binds to its receptor (uPAR) by its growth-factor-like domain, and initiates a proteolytic cascade at the surface of migrating cells to stimulate intracellular signaling responsible for cell migration and proliferation. The uPA lacking the growth-factor-like domain was, however, unable to associate with uPAR and was rapidly cleared from the cell surface (Poliakov et al., Biochem J., 2001, 355:639-45).

[0010] Binding of uPA to its receptor greatly potentiates plasminogen/plasmin conversion at the cell surface. Plasmin is a broadly specific serine protease, which can directly degrade components of the extracellular matrix. uPA and plasmin are somehow involved in cell morphogenesis by activating or inducing the release of morphogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), or fibroblast growth factor (FGF). Clinical observations correlate the presence of enhanced uPA activity at the invasive edge of the tumors (Schmitt M et al, Fibrinolysis, 1992, 6, 3-26). ATF is capable of mediating disruption of the uPA/uPAR complex and inhibiting tumor cell migration and invasion in vitro (H. Lu et al., FEBS Letter, 1994, 356, 56-59). However, the ATF molecule retains the EGF growth factor binding domain, which interacts with the uPAR receptor. Such interactions may facilitate tumor growth, as suggested in the scientific literature (Rabbani et al., J Biol. Chem 275:16450-58 (1992)).

SUMMARY OF THE INVENTION

[0011] The present invention provides kringle-containing polypeptides, called abrogens, that are potent inhibitors of endothelial proliferation and/or angiogenesis. The abrogen polypeptides are capable of inhibiting or reducing cell proliferation induced by both bFGF and VEGF in a specific endothelial cell proliferation assay, whereas angiostatin only inhibits bFGF induced proliferation in this assay. Furthermore, vectors that express abrogen polypeptides in vivo reduce tumor metastasis in two lung cancer models. Thus, aspects of the invention include novel polypeptides, nucleic acids that encode them, vectors containing them, and methods of using any of these aspects to express polypeptides, alter growth or other characteristics of cells, or treat or prevent disease.

[0012] Embodiments of the abrogen activity include a region of urokinase plasminogen activator encompassing the kringle domain. The mammalian urokinase plasminogen activator (uPA) kringle domain (ATF-kringle) has not been previously identified as a separate molecule with anti-angiogenic activity. Rather, it was previously shown to be a potent source of attraction of smooth muscle cells [2]. Surprisingly, the Applicant has identified and showed that the ATF-kringle retains a very potent anti-angiogenic activity, while not containing the growth-factor-like domain acting as binding site to the uPAR, thereby allowing uPA/uPAR complex disruption. As demonstrated in Example 3, for example, ATF-kringle containing polypeptides can inhibit endothelial cell activation and/or proliferation mediated by several different proangiogenic proteins, such as bFGF and VEGF, in a species independent manner.

[0013] The use of the kringle domain allows greater specificity in the anti-angiogenic mode of action. Our data from in vitro studies shows that the ATF-kringle molecule possesses a new activity that inhibits both bFGF and VEGF induced tube formation and/or cell proliferation in a specific endothelial cell assay. This assay also distinguishes the species-specific activity of other anti-angiogenic polypeptides. The abrogen polypeptides, and in particular those of SEQ ID No.: 1, 3, 5, 7, 9, and 10, do not show a species-specific response and both mouse and human derived polypeptides, for example, function in a mouse model system. This can be advantageous in developing human therapeutic compositions based upon a mouse model system. In another contrast over previous polypeptides, anti-angiogenic factors such as endostatin or angiostatin only inhibit bFGF-induced activity in this assay (Chen et al., Hum Gen Ther 11: 1983-96 (2000)). In general terms, the invention encompasses the production of, identification of, and use of polypeptides, as well as the nucleic acids that encode them, that possess this new activity, referred to as abrogens.

[0014] Thus, in one aspect, the invention comprises an isolated abrogen polypeptide, such as one with an amino acid sequence of SEQ ID NO.: 1, 3, 5, 7, 9, or 10 comprises repeated sequence thereof, between 2 to 5, and preferably between 3 to 4 repeated sequences of one or more of SEQ ID NO.: 1, 3, 5, 7, 9, or 10, the polypeptide being in a form that does not exist in nature and has not been previously disclosed. The abrogen polypeptide can be in purified form, so that, for example, it is no longer inside a cell that produces it, it is in an extract derived from a cell that produces it, it is at least partially separated from a final reaction mixture that produces it, or one or more components of a mixture containing it have been substantially or to a measurable extent removed. A purified form can also be a form suitable for pharmaceutical research use, such as a form substantially free of antigenic or inflammatory components. A purified form can also be the result of an affinity purification process.

[0015] The invention also includes a nucleic acid comprising or consisting of a sequence that encodes an abrogen polypeptide, such as the sequences of SEQ ID NO.: 2, 4, 6, or 8, or comprising repeated sequences thereof, between 2 to 5, and preferably between 3 to 4 repeated sequences of one or more of SEQ ID NO.: 2, 4, 6, or 8. The nucleic acid can be DNA, RNA, or DNA or RNA comprising modified nucleotide bases. A nucleic acid encoding an abrogen polypeptide can also be operably linked to a variety of one or more sequences used in expression vectors, and/or cloning vectors, and/or other vectors. For example, the abrogen encoding nucleic acid can be linked to a promoter, enhancer, a sequence encoding a signal sequence, and/or a sequence encoding an affinity purification sequence. One of ordinary skill in the art is familiar with selecting appropriate sequence(s) or vector(s) and using them. The invention also encompasses cells that contain or comprise an abrogen polypeptide or abrogen encoding nucleic acid.

[0016] The cell can be transduced with, transfected with, or have an introduced into it a vector that comprises the abrogen encoding nucleic acid. Progeny of the cell, for example cells that result from cultured cell splitting or maintenance procedures, are also included in the invention. The cell can be a cultured primary cell, an established cell line cell, a transformed cell, a tumor cell, an endothelial cell, or a variety of other mammalian cells.

[0017] The invention also comprises a novel purified polypeptide that comprises one or more fragments of a mammalian or human kringle-containing protein, and for example comprises a 2 to 5 repeated sequences and preferably 3 to 4 repeated sequences of one or more kringle fragments, the fragments having a kringle domain that is capable of inhibiting tube formation in endothelial cell cultures induced by bFGF and VEGF, and/or capable of reducing cell proliferation induced by bFGF and VEGF, and/or capable of inhibiting metastasis of mammalian tumors. This fragment does not contain an EGF-binding domain, such as the EGF-binding domain of uPA or the amino terminal fragment (ATF) of uPA. The novel purified polypeptide does not contain the exact amino acid sequence of the kringle 5 domain of human plasminogen, the exact sequence of kringle 2 from human prothrombin, the exact 80 amino acids beginning at residue 462 of human plasminogen, or the exact sequence of any of the previously disclosed kringle-containing polypeptides, peptides, or proteins. The polypeptide can comprise or consist of any one of SEQ ID NO: 1, 3, 5 or 7, or one or more of the sequences listed in FIG. 2, or repeated sequences thereof. The novel polypeptides can advantageously be used in a number of instances where inhibiting or reducing cell proliferation associated with bFGF and VEGF treatment is desired, and/or where inhibiting angiogenesis or tumor metastasis is desired.

[0018] In another aspect, the invention comprises a recombinant kringle-containing polypeptides consisting of 2 to 5, preferably 3 to 4 repeated kringle fragments.

[0019] In still another aspect, the invention comprises nucleic acids that encode these novel polypeptides, vectors containing them, and cells containing them. Preferably, inhibiting tube formation in endothelial cell cultures induced by bFGF and VEGF, reducing cell proliferation induced by bFGF and VEGF, and/or inhibiting metastasis of mammalian tumors is measured in culture with established endothelial cell lines or tumor cell lines. However, other types of measurements, including measurements in vivo, can also be used. In this and other aspects of the invention involving cells, a preferred embodiment employs or involves human umbilical vein endothelial cells or mammary or lung tumor cells.

[0020] Preferably, the kringle-containing protein is a human protein or fragment thereof, such as a human plasminogen activator, like urokinase plasminogen activator or tissue plasminogen activator. Other human proteins from which the novel polypeptides and nucleic acids of the invention can be derived are ApoArgC, Factor XII, hepatocyte growth factor activator, hyaluronan binding protein, macrophage stimulating protein, thrombin, retinoic acid receptors 1 and 2, and kringle-containing domains from extended sequence tag database or other databases. In preferred examples, these polypeptides comprise a kringle domain having a region of SEQ ID NO.: 1 from Asn 53 to Asp 59 [NYCRNPD], and further comprise one or more regions within a particular amino acid sequence identity range to a region of SEQ ID NO.: 1, 3, 5, or 7. In particular, the regions of SEQ ID NO.: 1 that may be modified include from Cys 3 to Trp 27, Asn 53 to Cys 84, Lys 1 to Thr 2, and Ala 85 to Asp 86. However, these derivatives contain the conserved 6 Cys residues that are thought to help properly fold the kringle domain into a characteristic structure. Various regions are quite amenable to modification by substitution, deletion, and/or addition, including the region from about Asn 28 to about His 52 or Lys 51, and the terminal 2 residues from each of the N terminus and C-terminus of SEQ ID NO.:1. Particularly preferred derivatives include those with a region of approximately 50% amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region of approximately 40% amino acid identity to the region of SEQ ID NO.: 1 from Asn 53 to Cys 84; a region of approximately 55% amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region of approximately 45% amino acid identity to the region of SEQ ID NO.: 1 from Asn 53 to Cys 84; a region of approximately 35% amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region of approximately 35% amino acid identity to the region of SEQ ID NO.: 1 from Asn 53 to Cys 84. For each of the abrogen regions identified here or elsewhere in this disclosure, one of skill in the art can clearly select an optimum or desirable range or specific sequence identity difference from that listed in the previous sentence. Thus, the 50% percent amino acid identity noted here and elsewhere can also be 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or from about 50-55%, or 55-60%, or 60-65%, or 65-70%, or 70-75%, or 75-80%, or 80-85%, or 85-90%, or 90-95%, or 95-98%, or 98-99%. Similarly, the 40% noted here or elsewhere can be 45%, 50%, and above and in various ranges as just listed, and the 35% noted here and elsewhere can be 40%, or 45% and above and in various ranges as just listed. Additional examples include an abrogen polypeptide with amino acid sequence of SEQ ID NO.: 1 modified to contain 1 to about 15 amino acid changes of substitutions, deletions, or additions, wherein the amino acid changes occur in the amino acids from Asn 28 to His 52, Lys 1 to Thr 2, Ala 85 to Asp 86. Furthermore, derivatives may merely contain or may additionally contain 1 to about 5, 1 to about 10, 1 to about 15, or 1 to about 20 amino acid changes outside of the consensus region from Asn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD] that are conservative amino acid substitutions.

[0021] The polypeptides and the nucleic acids that encode them may additionally have or encode a selected signal sequence region and/or an affinity purification sequence region. As used herein, the term “signal sequence or signal peptide” is understood to mean a peptide segment which directs the secretion of the abrogen polypeptides or abrogen fusion polypeptides and thereafter is cleaved following translation in the host cells. The signal sequence or signal peptide thus initiates transport of a protein across the membrane of the endoplasmic reticulum. Signal sequences have been well characterized in the art and are known typically to contain 16 to 30 amino acid residues, and may contain greater or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal peptide across the membrane lipid bilayer during transport of the nascent polypeptide. Following initiation, the signal peptide is usually cleaved within the lumen of the endoplasmic reticulum by cellular enzymes known as signal peptidases (von Heijne (1986) Nucleic Acids Res., 14: 4683). Numerous examples exist including the well known poly-His tag sequence, the immunoglobulin signal sequence, and the human interleukin 2 (IL2) signal sequence.

[0022] The polypeptide and the sequence encoding the polypeptide used in a specific vector encoding the given kringle domain may also be linked to stabilizing elements or polypeptides or the sequences that encode them, such as those from human serum albumin or the immunoglobulin Fc portion of an IgG molecule.

[0023] The abrogen polypeptides according to the present invention may be advantageously linked to one or more human serum albumin (HSA) protein sequences one or more other fusion partners. Such fusion polypeptides comprise the abrogen polypeptide fused at its C- or N-terminal, or both, with HSA. The amino acid sequence of HSA is well known in the art and is inter alia disclosed by Meloun et al. (Complete Amino Acid Sequence of HSA, FEBS Letter: 58:1. 136-137, 1975) and Behrens et al. (Structure of HSA, Fed. Proc. 34,591, 1975), and more recently by genetic analysis (Lawn et al., Nucleic Acids Research, 1981, 9, 6102-6114). Shorter forms or variants of HSA, as described in EP 322 094, may also be used to produce the abrogen fusion protein of the invention. Any abrogen polypeptide noted here can be used to prepare an abrogen fusion protein or polypeptide of the invention. Construction of such fusion proteins is well known in the art and is disclosed inter alia, in U.S. Pat. No. 5,876,969. Fusion proteins so obtained possess a particularly advantageous distribution in the body, while modifying the pharmacokinetic properties of the abrogen poplypeptide and compositions containing them, and favors the development of their biological activity.

[0024] An abrogen fusion protein or polypeptide according to the present invention may also comprise an N-terminal signal peptide, such as the IL2 signal peptide providing for secretion into the surrounding medium, followed or preceded by a HSA or a portion thereof, or a variant thereof and the sequence of the abrogen polypeptides. The abrogen polypeptides may be coupled either directly or via an artificial peptide or linker to albumin, at the N-terminal end or the C-terminal end or both.

[0025] The chimeric molecule may be produced by eucaryotic, prokaryotic, or cellular hosts that contain a nucleotide sequence encoding the abrogen fusion protein, and then harvesting the polypeptide produced. Animal cells, yeast, fungi may be used as eucaryotic hosts. In particular, yeasts of the genus of Saccharomyces, Kluveromyces, Pichia, Schwanniomyces, or Hansenula may be cited. Animal cells, such as for example, COS, CHO, 293 cell lines, and C127 cells, and the like may be used. Fungi such as Aspergillus sp., or Trichoderma ssp may be used. Bacteria, such as Esherichia coli, or bacteria belonging to the genera of Corynebacterium, Bacillus, or Streptomyces may be used as prokaryotic cells.

[0026] In another fusion protein or polypeptide example, the abrogen polypeptide is fused to one or more immunoglobulin Fc regions as described in WO 00/01133. Immunoglobulin Fc region is understood to mean the carboxylterminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region may comprise: 1) an immunoglobulin constant heavy 1 (CH1) domain, an immunoglobulin constant heavy 2 (CH2) domain, and an immunoglobulin constant heavy (CH3) domain; 2) a CH1 domain and a CH2 domain; 3) a CHI domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; and/or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment the Fc region used in the DNA construct encoding the abrogen polypeptide also enclodes an immunoglobulin hinge region, CH2 and CH3 domains, and depending upon the type of immunoglobulin used to generate the Fc region, optionally a CH4 domain. More preferably, the immunoglobulin Fc region comprises a hinge region, and CH2 and CH3 domains. Immunoglobulin from which the heavy chain constant region is preferably derived is IgG of subclasses 1, 2, 3, or 4, and most preferably of subclass 2, most preferably the murin or human immunoglobulin Fc region from IgG2a. Other classes of immunoglobulin, IgA, IgD, IgE and IgM, may be used. The choice of appropriate or advantageous immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art. The Fc region used in the fusion protein is preferably from a mammalian species, for example from murine origin, and preferably from human origin, or from a humanized Fc region.

[0027] The fusion proteins of the invention preferably are generated by conventional recombinant DNA methodologies. The fusion proteins preferably are produced by expression in a host cell of a DNA molecule encoding a signal sequence, an immunoglobulin Fc region or HSA for example, and an abrogen polypeptide. The constructs may encode in a 5′ to 3′ direction, the signal sequence, the immunoglobulin Fc region or HSA for example, and the abrogen polypeptide. Alternatively, the constructs may encode in a 5′ to 3′ direction, the signal sequence, the abrogen polypeptide and the immunoglobulin Fc region or HSA for example. As noted above, other fusion partners or stabilizing elements or polypeptides can be selected for use. The abrogen polypeptide may be coupled either directly or via a linker to the immunoglobulin Fc region or HSA, for example. The fusion of the abrogen with the immunoglobulin Fc region are produced by introducing into mammalian cell such constructs, and culturing the mammalian cells to produce the fusion proteins. The resulting fusion protein can be harvested, refolded if necessary, and purified using conventional purification techniques well known and used in the art. The resulting abrogen polypeptides exhibit longer serum half-lives, presumably due to their larger molecular sizes, and other advantageous properties.

[0028] The abrogen polypeptides and either the HSA or the immunoglobulin Fc region, for example, may be linked by a polypeptide linker. As used herein the term “polypeptide linker” is understood to mean a peptide sequence that can be used to link two proteins together or a protein and an Fc region. The polypeptide linker preferably comprises a plurality of amino acids such as glycine and/or serine. Preferably, the polypeptide linker comprises a series of glycine and serine peptides about 10-15 residues in length. See, for example, U.S. Pat. No. 5,258,698, the disclosure of which is incorporated herein by reference. More preferably, the linker sequence is as set forth in SEQ ID NO: 12 or 16, or comprises an Asp-Ala or an Arg-Leu sequence. It is contemplated however, that the optimal linker length and amino acid composition may be determined by routine experimentation.

[0029] The present invention also provides methods for producing abrogen from non-human species and as fusion proteins, such as with HSA and Fc regions. Non-human angiogenesis inhibitor fusion proteins are useful for preclinical studies of angiogenesis inhibitors because efficacy and toxicity studies of a protein drug must be performed in animal model systems before testing in humans. A human protein may elicit an immune response in mouse, and/or exhibit different pharmacokinetics, skewing the test results. Therefore, the equivalent mouse protein is the best surrogate for the human protein for testing in a mouse model.

[0030] Additionally, various promoter/enhancer and RNA transcript stabilizing elements may be included in the vector.

[0031] In another aspect, the invention comprises methods for analyzing or identifying a polypeptide that reduces or inhibits endothelial cell proliferation induced by bFGF and VEGF, and/or reduces or inhibits tube formation induced by bFGF and VEGF, and/or reduces or inhibits tumor metastasis. In general, the method may comprise selecting a polypeptide having a kringle domain from a mammalian protein, the kringle domain comprising amino acid residues Asn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD], the kringle domain also containing 6 Cys residues and 2 Trp residues, and introducing the polypeptide to an endothelial cell, for example by employing an expression vector such as a recombinant adenoviral vector, a recombinant adeno-associated viral vector, or a plasmid vector. Any method for measuring the relative inhibition of tubule formation, the relative inhibition of cell proliferation, or the relative inhibition of tumor metastasis can be employed to detect a polypeptide having the appropriate characteristic or even a combination of characteristics. The invention specifically includes polypeptides and nucleic acids encoding these polypeptides that are identified or are capable of being identified by these methods.

[0032] Moreover, an abrogen polypeptide and compositions comprising it may be used as a therapeutic. The polypeptide and the method for expressing it in a cell can be, therefore, used in methods to treat or prevent a variety of angiogenesis related diseases or conditions, including, but not limited to hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis, diabetic neovascularization, diabetic retinopathy, macular degeneration, wound healing, obesity, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, psiorasis, ovulation-related disorders, menstruation-related disorders, placentation, psoriasis, and cat scratch fever.

[0033] In general, the use can also be for abrogating tumor vasculature growth or angiogenesis associated with a tumor. One skilled in the art is familiar with polypeptide expression and purification systems as well as methods for administering polypeptides and vectors in appropriate pharmaceutical compositions.

[0034] In addition, combination treatments where one or more angiogenesis inhibitor polypeptides of the invention, or abrogen polypeptides, are administered with one or more therapeutic compounds, polypeptides, or proteins. Also, the abrogen polypeptides or fusion proteins thereof can also be used in combination with the use of other therapeutic agents and in a combination with multiple, different abrogen or fusion polypeptides. Any existing or available therapeutic treatments can be combined with the polypeptides, combinations, or methods described here. Numerous examples exist and the compounds and the treatment methods can be selected from those available, such as those in the Physician's Desk Reference, Remington's Pharmaceutical Sciences, or Remington's Science and Practice of Pharmacy. A combination with an erythropoietin is specifically noted. Combinations with treatments or compounds that implicate angiogenesis or anti-angiogenesis mechanisms are preferred, but other tumor suppressing treatments and anti-cancer treatments or treatments used in cancer patients can also be selected.

[0035] In another aspect, the nucleic acids encoding an abrogen polypeptide can be used in a gene transfer method. The examples show how recombinant plasmid and adenoviral vectors, for example, can be used to affect metastasis in a lung tumor model. Various gene transfer and gene therapy vectors can be used in conjunction with the nucleic acids of the invention to either analyze the activity of an abrogen polypeptide in vivo or treat, prevent, or ameliorate an angiogenesis-related disease or condition in an animal. Preferably, the animal is human or mouse. More particularly, a nucleic acid encoding an abrogen of SEQ ID NO.: 1, 3, 5, 7, 9, or 10 or a repeat thereof, can be cloned into a vector, preferably an adenoviral vector, an adeno-associated virus (AAV), a plasmid, or other suitable viral or non-viral vector. In one embodiment, the vector is administered to a tumor bearing or non-tumor bearing animal by direct intratumoral injection, intravenous injection, intramuscular injection, electrotransfer-mediated administration, or other suitable method. The efficacy of the abrogen expressed from the vector can be assessed in the context of, for example, reduction of the primary tumor and/or abrogation of metastatic dissemination.

[0036] Accordingly, the invention comprises gene transfer methods and methods for expressing abrogen polypeptides in a cell of an animal. These methods may comprise inserting a selected abrogen encoding sequence, such as one encoding SEQ ID NO.: 1, 3, 5, 7, 9, 10 or one selected from FIG. 2, into a mammalian expression vector or the expression cassette of an appropriate vector. The vector is administered to a cell of the animal by any number of methods available, including intratumoral injection, electrotransfer, infusion, subcutaneous injection, intramuscular injection, or intravenous administration. The effect of the expressed abrogen polypeptide can then be measured and compared to control. These methods can be used to treat any one of a number of angiogenesis related diseases or disorders, such as those listed above.

[0037] While the production of kringle-containing polypeptides has been previously discussed, the successful and efficient production of soluble forms of biologically active abrogen polypeptides from E. coli has not. An aspect of the invention, therefore, is the use of expression vectors and fusion protein constructs to efficiently produce soluble abrogen polypeptides from E. coli. A related aspect of the invention is the novel constructs and vectors that encode abrogen polypeptides and fusion proteins of abrogen polypeptides that can be used to express soluble abrogen polypeptides and fusions from E. coli. Advantageously, the methods, vectors, and constructs described and exemplified produce comparatively high levels of soluble fusion protein per gram of wet cell pellet. Furthermore, the ability to directly express measurable or high levels of soluble fusion protein from E. coli simplifies the purification and production of protein.

[0038] It is known that peptides and proteins may be produced via recombinant means in a variety of expression systems, such as various strains of bacterial, fungal, mammalian or insect cells. The production of small heterologous peptides recombinantly for effective research and therapeutic use encounters however several difficulties. They may be for example subject to intracellular degradation by proteases and peptidases present in the host cell. In particular, it has been previously reported that the various kringles of human plasminogen, i.e, kringles 2 (Eur. J. Biochem. 1994 219 p455), or kringle 3 (Eur. J. Biochem. 1994 219 p 455) or kringles 2 and 3 (Biochemistry 1996 35 p2357), or again kringle 4 (Biochemistry 2000 39 p7414-7419), are unable to adopt a stable soluble conformation when produced in E. coli. Therefore, these kringle polypeptides are generally accumulated and are found in the insoluble or “inclusion bodies” fraction, which render them almost useless for screening purposes in biological or biochemical assays. Furthermore, these inclusion bodies usually require further manipulations in order to solublize and properly refold the heterologous proteins. These additional steps are technically difficult and expensive and essential render useless any high throughput applications or project, that is for the practical production of recombinant proteins for therapeutic, diagnostic or other research use.

[0039] Several different fusion protein partners with a desired heterologous peptide to protein are proposed in the art to enable the recombinant expression and or secretion of an heterologous protein. These fusions protein include inter alia LacZ, trpE fusion proteins, maltose binding protein fusions (MBP, Bedouelle et al., Eur.J.Biochem, 1988, 171(3): 541-9), the glutathione-S-transferase fusion protein (GST, Smith et al., Gene, 1988, 67(1): 31-40), the Z domain from the protein A (Z, Nilson et al., Protein Eng., 1987, 1:107-113), thioredoxin (TrxA, La Vallie et al., Biotechnology, 1993, 11: 187-193; Hoog et al., Biosci. Rep. 4:917, 1984), NusA (Davis et al., Biotechnol. Bioeng., 1999, 65: 382-388), and the Gb-i domain from the protein G (Gb1, Huth et al., Protein Sci., 1997, 6:2359-64), at the amino- or the carboxy- termini.

[0040] In this regard, Hammarstrom et al. (Protein Science 11:313 (2002) provides some discussion as to the effect of different fusions, namely GST, NusA, ZZ (double Z domain of protein A), Gb1, MBP, and TrxA, upon expression and solubilization of 32 potentially interesting human proteins having various characteristics in terms of size, cysteine content, and their solubility probability. While none appear outstanding, MBP seems to be somewhat better than the other fusion partners.

[0041] Kapust et al. (Protein Science 8:1668, 1999) also compared three soluble fusion partners MBP, TrxA, and GST to inhibit aggregation of six diverse proteins that normally accumulate in an insoluble form, and reports that MBP is far more effective for solubilizing than the two other partners, in that the MBP fusion partners invariably proved to be more soluble than GST and TrxA, and thus rendered the protein capable of adopting a stably folded conformation.

[0042] However, neither Hammarstrom et al. nor Kapust et al. have specifically addressed the problem of solubility of peptides having a cysteine content of around at least 7% and post-translational modifications such as the formation of disulfide bonds, although this refolding can be critical to produce or retain the activity of the protein.

[0043] The Applicant has now discovered and shown that among the existing fusion partners the thioredoxin (TrxA) is in fact capable of providing a very advantageous effect in terms of solubility of proteins having a cysteine content of around 7% and comprising 3 disulfide bonds, such as the abrogen polypeptides. This superior result was unexpected, as the previously existing guidelines have been found to be only partially predictable for producing stable, soluble and biologically active protein forms.

[0044] The invention thus comprises a method for producing a soluble abrogen polypeptide that comprises preparing a nucleic acid fusion construct comprising at least a TrxA encoding sequence fused in frame to an abrogen polypeptide sequence, as for example any one of SEQ ID NO: 1, 3, 5, 7, 9, 10, or those listed in FIG. 2. As in other aspects of the invention, the fusion partner encoding sequence can be located at the N-teminus, the C-terminus, or both ends of the abrogen encoding sequence, and different combinations of fusion partners can be selected for use. Preferably, the TrxA fusion partner is fused to the N-terminal of the abrogen. The amino acid sequence of the TrxA fusion partner is provided in SEQ ID NO: 22. The TrxA-abrogen fusion according to the invention may further comprise a linker peptide between the TrxA sequence and the abrogen sequence, which advantageously provides a selected cleavage site. Preferred cleavage site used is a thrombin cleavage site comprising the following amino acid sequence LVPRGS (SEQ ID NO: 23).

[0045] The present invention thus provides for an efficient method of increasing solubility of recombinant abrogen peptides. The abrogen produced by the method according to the present invention is obtained in an unexpected highly soluble form. The fusion protein is cytoplasmic and can be easily recovered by lysing the bacteria or host cell, purified and cleaved using for example the thrombin cleavage site. The nucleic acid construct can be incorporated into a vector or otherwise manipulated into a cell in order to express the fusion abrogen polypeptide. To produce the TrxA-abrogen fusion protein of this invention, a host cell is either transformed with, or has integrated in its genome, a DNA molecule comprising the TrxA-abrogen fusion protein, preferably under the control of an expression control sequence capable of directing the expression of the fusion protein production. Any one of a number of available expression control sequences can be selected for use. In preferred embodiments, the expression control sequences can operate in bacterial cells, such as E. coli, in order to express soluble fusion protein in E. coli cultures or cells.

[0046] Host cells suitable for the present invention are preferably bacterial cells, such as the various strains of E.coli, which are well known host cells in the field of biotechnology. The E.coli strain BL21 lambda DE3, used in the Examples, is preferably used, and most preferably the E.coli BL21 lambda DE3 trxB⁻ (Novagen), which has a mutation in the thioredoxine reductase (trxB gene) is used, thereby allowing for the fonnation of disulfide bond in E.coli cytoplasm.

[0047] The trxA-abrogen fusion protein may be purified by conventional procedures including selective precipitation solubilization and column chromatography methods. Preferably, a purification tag is included between the trxA and the abrogen sequence, eventually in upstream or downstream position of the cleavage proteolytic site for the thrombin (SEQ ID NO: 23). Purification tag sequences are well known in the art and include inter alia Arg-tag, calmodulin-binding peptide, cellulose binding domain, DsbA, c-myc-tag, FLAG-tag, HAT-tag, HIS-tag, and Strep-tag (Terpe K., Appl. Microbiol. Biotechnol, 2003, 60(5): 523-33). Preferably, the purification tags, such as a His tag sequence, which comprises 6 histidine residues, and the streptokinase tag comprising a nine-amino acid peptide having intrinsic streptavidin binding activity, such as for examples the sequences AWRHPQFGG or WSHPQFEK (Lamla et al., Mol.Cell.Proteomics, 2002, 1(6): 466-71) are used or incorporated into the fusion protein construct or encoded by the vector. One or more cleavage sites to liberate abrogen polypeptide from the fusion protein can also be used in the fusion protein construct or encoded by the vector.

[0048] The invention also comprises administration of one or more recombinant abrogen polypeptides in a cell of an animal. These methods may comprise administering the abrogen peptide as in SEQ ID NO: 1, 3, 5, 7, 9, or 10, (plus the peptide produced in E. coli with 4 extra amino acids at the N terminal sequence as listed in Example 12) or an abrogen fusion construct thereof as in SEQ ID NO: 13, 14, 15, 17, 18, 20, or 21, by any well-known method in the art, including for example, direct injections of the peptide at a specific site, i.e., by ophthalmic (including intravitreal or intraorbital), intraperitoneal, intramuscular, or intratumoral injections.

[0049] The invention also includes compositions comprising the abrogen polypeptides or nucleic acids, and the derivatives and nucleic acids encoding derivatives, such as those having the sequences of sequences listed herein or an abrogen fusion construct thereof as in SEQ ID NO: 13, 14, 15, 17, 18, 20, or 21, or nucleic acids encoding them. The abrogen polypeptides or derivatives can be recombinant polypeptides or purified polypeptides. The compositions of the present invention may be provided to an animal by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally). Where the composition is to be provided parenterally, such as by intravenous, subcutaneous, ophthalmic (including intravitreal or intracameral), intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracistemal, intracapsular, intranasal or by aerosol administration, the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution. Thus, the carrier or vehicle is physiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance. The fluid medium for the agent thus can comprise normal physiologic saline (e.g., 9.85% aqueous NaCl, 0.15 M, pH 7-7.4). In one embodiment, the composition is a pharmaceutically acceptable composition. One skilled in the art is familiar with selecting and testing pharmaceutically acceptable compositions for use with recombinant polypeptides and nucleic acids.

[0050] The abrogen formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s).

[0051] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0052] It is another aspect or object of the present invention to provide a method of treating diseases and processes that are mediated by angiogenesis.

[0053] It is yet another aspect of the present invention to provide a method and composition for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis, diabetic neovascularization, diabetic retinopathy, macular degeneration, wound healing, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, placentation, psoriasis, obesity and cat scratch fever.

[0054] It is another aspect of the present invention to provide a composition for treating cancer or repressing the growth of a cancer.

[0055] It is still another aspect of the present invention to provide a method for treating ocular angiogenesis related diseases such as macular degeneration or diabetic retinopathy by direct ophthalmic injections of the recombinant abrogen peptides.

[0056] Another aspect of the present invention is to provide a method for targeted delivery of abrogen compositions to specific locations.

[0057] Yet another aspect of the invention is to provide compositions and methods useful for gene therapy for the modulation of angiogenic processes.

[0058] Throughout this disclosure, applicants refer to journal articles, patent documents, published references, web pages, sequence information available in databases, and other sources of information. One skilled in the art can use the entire contents of any of the cited sources of information to make and use aspects of this invention. Each and every cited source of information is specifically incorporated herein by reference in its entirety. Portions of these sources may be included in this document as allowed or required. However, the meaning of any term or phrase specifically defined or explained in this disclosure shall not be modified by the content of any of the sources. The description and examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.

BRIEF DESCRIPTION OF THE FIGURES

[0059]FIG. 1: The proliferative response of transduced HUVEC human endothelial cells to human abrogen (hATF-K; SEQ ID NO. 1) and mouse abrogen (mATK-K; SEQ ID NO.: 3). Cultured cells were transduced with adenoviral vectors containing an expression cassette for producing the abrogen polypeptide (hATF-K and mATF-K), a control, CMV promoter only vector (CMV), and the full amino terminal fragment of plasminogen (hATF or mATF). In FIG. 1A, the left axis indicates the degree of cell proliferation and each of the boxes represents the level of cell proliferation under a treatment regimen as indicated by the addition of bFGF, VEGF, or both. The reduction in cell proliferation in all samples where the human abrogen polypeptide is expressed (hATF-K) is markedly reduced compared to controls (CMV, hATF, and mATF). The proliferation in the mouse abrogen expressing cells (mATF-K) is also markedly reduced. FIG. 1B shows representative cell cultures from mouse and human full ATF polypeptides and mouse and human ATF-Kringle containing abrogen polypeptides (see Examples). The first page shows Control (full human ATF treated with FGF) compared to hATF-Kringle containing polypeptide treated with FGF. The remaining pages list the adenoviral vector used to transduce the cells (see Examples).

[0060]FIG. 2: Various human protein sequences having a kringle domain possessing the consensus region from Asn 53 to Asp 59 of SEQ ID NO.: 1 and the with the 6 conserved Cys, 2 conserved Trp, and conserved Gly and Arg residues aligned. These proteins and homologs, isoforms, and derivatives of them, can be used in methods of the invention and used to produce polypeptides and polynucleotides of the invention.

[0061]FIG. 3: Effect of anti-angiogenic polypeptides on tubule growth in endothelial cells.

[0062] Because culture conditions rapidly deplete anti-angiogenic factors if they are added as a recombinant or purified polypeptide, HUVECs are directly transduced with adenoviral vectors to provide consistent protein expression and secretion for the duration of the assay (7-10 days). HUVECs are transduced with Adenovirus expressing: human abrogen, hATF-K (as in SEQ ID NO.: 1), mouse abrogen, mATF-K (as in SEQ ID NO.: 3), and human endostatin (FIG. 3A) or human Angiostatin (FIG. 3B). Control adenovirus containing the LacZ or no gene of interest (empty control) is also included. The transduced cells are then cultured in a 3-dimensional matrix of fibrin with recombinant VEGF or bFGF added, as indicated. Tubule formation as a marker for activation and proliferation of endothelial cells is then visualized and recorded. Tubule formation in both the bFGF and VEGF treated cells is markedly inhibited in only the abrogen expressing cultures.

[0063]FIG. 4: Prevention of tumor metastasis in mouse 4T1 lung cancer model. Control empty plasmid and abrogen (hATF-K or mATF-K) expression cassette containing plasmid introduced via electrotransfer 6 days prior to injection of 4T1 tumor cells. Approximately 250,000 tumor cells are injected subcutaneously. Fifteen days after injection, primary tumors are removed in a surgical procedure. Lungs are harvested 35 days post tumor injection and the size and number of metastatic tumor colonies measured.

[0064]FIG. 5: Prevention of tumor metastasis in mouse 4T1 lung cancer model. Control empty plasmid compared to mATF-K expression plasmid. The assay protocol is the same as in FIG. 4.

[0065]FIG. 6: Prevention of tumor metastasis in mouse 3LL Boston lung cancer model. Control empty plasmid compared to mATF-K expression plasmid. The assay protocol is the same as FIG. 4, with the exception that 3LL Boston cells are used.

[0066]FIG. 7: Prevention of tumor metastasis in mouse 3LL Boston lung cancer model. Control empty plasmid compared to experimental control mEndostatin expression plasmid. The assay protocol is the same as FIG. 6.

[0067]FIG. 8: Measurement of size and number of metastasis in the 4T1 lung tumor model described for FIG. 4. Each spot represents the weight of the lung from each animal surveyed (C57BL/6 mice), indicating the relative size of the tumor nodules present. The left axis indicates the number of visible tumor nodules for each of the animals. With the exception of one animal in the hATF-K sample, the abrogen expressing vector treatment animals show a reduction in both the size and number of metastatic tumor nodules as compared to control. The hATF-K animals with abnormally high number of nodules were not further examined for experimental or procedural error or expression of hATF-K. Here the controls are empty plasmid (Control) and an alkaline phosphatase expressing control plasmid (mSEAP).

[0068]FIG. 9: Measurement of size and number of metastasis in the 3LL Boston lung tumor model described for FIG. 4 using the graphical representation method described for FIG. 7. Controls are the same as in FIG. 7. Again, the use of both the mouse and human abrogen expressing vectors (mATF-K and hATF-K) results in significant reduction in tumor metastasis.

[0069]FIG. 10: Measurement of size and number of metastasis in the 3LL Boston lung tumor model as described for FIG. 9. These data indicate that treatment with mouse endostatin or angiostatin, or either mouse or human ATF-K, reduce the number and size of the lung metastatic nodules compared to control treatment. The fact that both mouse and human abrogen encoding vectors are efficacious indicates that the species-specific characteristics that limit the use of the endostatin and angiostatin polypeptides are not present in the abrogen polypeptides. Furthermore, the abrogen polypeptides appear at least as efficacious as the either endostatin or angiostatin and much more efficacious than a combined endostatin/angiostatin treatment (mEndo/mAngio).

[0070]FIG. 11: Systemic expression of mouse or human derived abrogen polypeptides (here listed as MuPAK or HuPAK) from vector introduced into muscle significantly reduces the formation of spontaneous lung metastases in the 3LL-B tumor model. Systemic expression of therapeutic transgenes from the muscle is established 6 days before C57BL/6 mice are injected with a tumorigenic dose of 3LL-B tumor cells. The primary tumor is carefully excised 15 days post cell injection. The study is terminated on day 35 and lung metastases were counted. Panel A: lungs from mice treated with empty expression vector; Panel B: mice treated with human derived ATF-Kringle abrogen expressing vector (HuPAK); and Panel C: with treated with mouse derived ATF-Kringle abrogen expressing vector (MuPAK); Panel D: graphically shows the number and size of metastatic nodules present as the diameter of each “bubble” represents the lung weight.

[0071]FIG. 12: Systemic expression of mouse or human abrogen (here listed as MuPAK or HUPAK) from muscle significantly reduces the formation of spontaneous lung metastases in the MDA-MB-435 tumor model. Systemic expression of therapeutic transgenes from the muscle is established 10 days after SCID/bg mice are injected with a tumorigenic dose of MDA-MB-435 (human breast adenocarcinoma tumor cells). The primary tumor is carefully excised when a volume of 250 to 350 mm3 is reached. The study is terminated on day 89 and lung metastases measured. Panel A: lungs from mice treated with control mSEAP; Panel B: with treated with mouse derived ATF-Kringle abrogen expressing vector (here MuPAK); Panel C: mice treated with human derived ATF-Kringle abrogen expressing vector (HuPAK); and Panel D: graphically shows lung metastases counts as noted above.

[0072]FIG. 13A: is a schematic representation of the plasmid pXL2996.

[0073]FIG. 13B: is a schematic representation of the plasmid pMB063.

[0074]FIG. 13C is a schematic representation of the plasmid pBA140.

[0075]FIG. 14: is a schematic representation of the plasmid pMB060 and fusion construct.

[0076]FIG. 15: is a schematic representation of the plasmid pMB059 and fusion construct.

[0077]FIG. 16 is a schematic representation of the plasmid pMB056 and fusion construct.

[0078]FIG. 17: is a schematic representation of the plasmid pMB055 and fusion construct.

[0079]FIG. 18: is a schematic representation of the plasmid pMB060m prepro and fusion construct.

[0080]FIG. 19: is a schematic representation of the plasmid pMB053 and fusion construct.

[0081]FIG. 20: is a schematic representation of the plasmid pMB057 and fusion construct.

[0082]FIG. 21: is a schematic representation of the plasmid pXL4128.

[0083]FIG. 22: is a schematic representation of the plasmid pET28-Trx, which can be used in the methods to produce abrogen fusion protein.

[0084]FIG. 23: is a schematic representation of plasmids pXL4189 (top) and pXL4215 (bottom).

[0085]FIG. 24: is a schematic representation of plasmids pXL4190 (top) and pXL4219 (bottom).

[0086]FIG. 25: Production of Fusion Proteins. This Figure shows the expression products from various plasmids separated by gel electrophoresis. The far left lane of the gel image (lane #M) shows the molecular weight markers, indicated by the numbers on the left side (Kda). Lane #2 is the total cell extract from cell expression using pXL4189 (TrxA-abrogen N43 fusion), for expressing abrogen N43. Lane #8 is the soluble fraction from the cell expression of Lane #2. The results show that a substantial percentage of fusion protein is soluble and can be cleaved to produce soluble abrogen N43. Lane #9 is the remaining cell pellet from Lane #2. Lane #5 is the total cell extract from cell expression using pXL4190 (TrxA-K4 angiostatin fusion), for expressing K4 kringle domain from angiostatin. Lane #10 is the soluble fraction from the cell expression of Lane #5. The results show that a substantial percentage of fusion protein is soluble and can be cleaved to produce soluble K4 polypeptide. Lane #11 is the remaining cell pellet from Lane #5.

[0087]FIG. 26: Analysis of Purified Abrogen by SDS-PAGE. This Figure shows the production and purification results from an abrogen expression and cleavage method as described in Example 12. The various levels of protein loaded on the gel indicate the purity of the abrogen D43 polypeptide, that the fusion protein is no longer present, and that no other protein components are visible.

[0088]FIG. 27: Biological effect of recombinant kringle domains produced in E. coli on tubule growth in HUVE cell spheroids. Recombinant kringle domains are added to HUVEC spheroids in a 3-dimensional matrix of fibrin containing VEGF and bFGF. Tubule formation is then visualized and recorded at day 11 post treatment with the test product. Tubule formation in the bFGF and VEGF treated cells is markedly inhibited only in the presence abrogen D43 and plasminogen K5 kringle domain.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0089] A number of Kringle domain containing proteins and polypeptides have been described and used in a variety of methods, including therapeutic methods. As shown here, a kringle-containing abrogen polypeptide can be identified and used to inhibit or reduce tumor metastasis, inhibit or reduce endothelial cell proliferation, and/or inhibit or reduce endothelial cell tubule formation. As an abrogen polypeptide or nucleic acid encoding an abrogen polypeptide, specific examples include the mouse or human derived kringle domains of uPA (SEQ ID NO.: 1-8). Additional examples have been mentioned and/or are described below in their structure and/or method of making and identifying. Functionally, an abrogen polypeptide can be distinguished by the ability to inhibit tumor metastasis. A more specific set of abrogen polypeptides include those that inhibit the endothelial cell proliferation induced by both of bFGF and VEGF, either in separate assays or together in one assay. An abrogen polypeptide can be either secreted or expressed inside a cell.

[0090] In making and using aspects and embodiments of this invention, one skilled in the art may employ conventional molecular biology, cell biology, virology, microbiology, and recombinant DNA techniques. Exemplary techniques are explained fully in the literature. For example, one may rely on the following general texts to make and use the invention: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Sambrook et al., Third Edition (2001); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation, Hames & Higgins, eds. (1984); Animal Cell Culture (RI. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); Gennaro et al. (eds.) Remington's Pharmaceutical Sciences, 18th edition; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (2001), Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, Inc. (2001); W. Paul et al. (eds.) Fundamental Immunology, Raven Press; E. J. Murray et al. (ed.) Methods in Molecular Biology: Gene Transfer and Expression Protocols, The Humana Press Inc. (1991); J. E. Celis et al., Cell Biology: A Laboratory Handbook, Academic Press (1994); J. E. Coligan et al. (Eds.) Current Protocols in Protein Science, John Wiley & Sons (2001); and J. S. Bonifacino et al. (Eds.) Current Protocols in Cell Biology, John Wiley & Sons, Inc. (2001). Additional information sources are listed below or are referred to by citation number corresponding to the references at the end of the specification.

[0091] As used herein, a “vector” means any nucleic acid or nucleic acid-bearing particle, cell, or organism capable of being used to transfer a nucleic acid into a host cell and/or used to cause the expression of a polypeptide in a host cell. The term “vector” includes both viral and nonviral products and means for introducing the nucleic acid into a cell. A “vector” can be used in vitro, ex vivo, or in viva. Non-viral vectors include plasmids, cosmids, and can comprise liposomes, electrically charged lipids (cytofectins), DNA protein complexes, and biopolymers, for example. Viral vectors include retroviruses, lentiviruses, adeno-associated virus, pox viruses, baculovirus, reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, and adenovirus vectors, for example. Vectors can also comprise the entire genome sequence or recombinant genome sequence of a virus. A vector can also comprise a portion of the genome that comprises the functional sequences for production of a virus capable of infecting, entering, or being introduced to a cell to deliver nucleic acid therein

[0092] The abrogen derivatives of this invention include those having one or more conservative amino acid substitutions. For example, one or more amino acid residues within a sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent when the substitution results in no significant change in activity in at least one selected biological activity or function.

[0093] Substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0094] “Isolated,” when referring to a nucleic acid or polypeptide, means that the indicated molecule is present in the substantial absence of at least one other molecule with which it naturally occurs or necessarily occurs because of its method of preparation. Thus, for example, an “isolated abrogen polypeptide” refers to a molecule substantially free of a macromolecule existing in a cell used to produce the abrogen polypeptide. However, the preparation or sample containing the molecule may include other components of different types. In addition, “isolated from” a particular molecule may also mean that a particular molecule is substantially absent from a preparation or sample. Varying degrees of isolation can be prepared from methods known in the art. Similarly, a “purified” form of a molecule is at least partially separated from a final reaction mixture that produces it, or one or more components of a mixture containing it have been substantially or to a measurable extent removed. A purified form can also be a form suitable for pharmaceutical research use, such as a form substantially free of antigenic or inflammatory components. A purified form can also be the result of an affinity purification process or any other purification step or process.

[0095] The “derivatives” noted here can be produced using homologue sequences, modifications of an existing sequence, or a combination of the two. The term “homologue” is used herein to refer to similar or homologous sequences, whether or not any particular position or residue is identical to or different from the molecule similarity or homology is measured against. A nucleic acid or amino acid sequence alignment may include spaces. Preferably, alignment is made using the consensus residues listed in FIG. 2, or the 6 Cys residues of the kringle domain. One way of defining a homologue is through “percent identity” between two nucleic acids or two polypeptide molecules. This refers to the percent defined by a comparison using a basic blastn or blastp or blastx algorithm at the default setting, unless otherwise indicated (see, for example, NCBI BLAST home page: http://www.ncbi.nlm.nih.gov/BLAST/). Aligning a Cys residue in abrogen, for example, can be performed by comparing sequences where the first amino acid residue or codon is for a particular Cys, or where the particular Cys residue is set at the same position as that of the abrogen Cys residue. For example, the blastp algorithm was used to generate homologue sequences, as in those of FIG. 2, by selecting the Blosum62 matrix, gap costs set at Existence: 11 and Extension: 1 (the default settings when performed). Typically, the default setting is used unless otherwise indicated. “Homology” can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions allowing for the formation of stable duplexes between homologous regions and determining of identifying double-stranded nucleic acid.

[0096] A “functional homologue” or a “functional equivalent” of a given polypeptide or sequence includes molecules derived from the native polypeptide sequence, as well as recombinantly produced or chemically synthesized polypeptides, which function in a manner similar to the reference molecule or achieve a similar desired result. Thus, a “functional homologue” or a “functional equivalent” of a given kringle nucleotide region includes similar regions derived from a different species, nucleotide regions derived from an isoform, or from a different cellular source, or resulting from an alternative splicing event, as well as recombinantly produced or chemically synthesized nucleic acids that function in a manner similar to the reference nucleic acid region in achieving a desired result, such as a result in a particular assay or cell characteristic.

[0097] A “recombinant” molecule is one that has undergone at least one molecular biological manipulation, as known in the art. Typically, this manipulation occurs in vitro but it can also occur within a cell, as with homologous recombination. A recombinant polypeptide is one that is produced from a recombinant DNA or nucleic acid. A “coding sequence” or “sequence that encodes” is a sequence capable of being transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′(carboxyl) terminus.

[0098] A “nucleic acid” is a polymeric compound comprised of covalently linked nucleotides, from whatever source. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or doublestranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The term “nucleic acid” also captures sequences that include any of the known base analogues of DNA and RNA.

[0099] A cell has been “transfected” by a vector or exogenous or heterologous nucleic acid when the vector or nucleic acid has been introduced inside the cell. A cell has been “transformed” or “transduced” by a vector or exogenous or heterologous nucleic acid when the vector or nucleic acid effects a phenotypic change or detectable modification in the cell, such as expression of a polypeptide.

[0100] Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 7:980-990 (1992)). Preferably, the viral vectors are replication defective or conditionally replication defective, that is, they are unable to replicate autonomously in the target cell or unable to replicate autonomously under certain conditions. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome necessary for encapsulating the viral particles.

[0101] DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330 (1991)), defective herpes virus vector lacking a glyco-protein L gene, or other defective herpes virus vectors (PCT Publication WO 94/21807 and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626-630 (1992); see also La Salle et al., Science 259:988-990 (1993)); a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101 (1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski et al., Mol. Cell. Biol. 8:3988-3996 (1988)); and a conditional replicative recombinant vectors (see, for example, U.S. Pat. Nos. 6,111,243, 5,972,706, and published PCT documents WO 00136650, WO 0024408).

[0102] Recombinant adenoviruses display many advantages for use as transgene expression systems, including a tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (see e.g., Berkner, K. L., Curr. Top. Micro. Immunol., 158:39-66 (1992); Jolly D., Cancer Gene Therapy, 1:51-64 (1994)).

[0103] It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter or eletrotransfer device (see, e.g., Wu et al., J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)). Naked plasmids or cosmids can be used in a number of gene transfer protocols and these plasmids and cosmids can be used in embodiments of this invention (see, in general, Miyake et al., PNAS 93:1320-1324 (1996); U.S. Pat. No. 6,143,530; U.S. Pat. No. 6,153,597; Ding et al., Cancer Res., 61:526-31 (2001); and Crouzet et al., PNAS 94:1414-1419 (1997). Among the preferred plamid vectors are those described in WO9710343 and WO9626270. Plasmids can also be combined with lipid compositions, pharmaceutically acceptable vehicles, and used with electrotransfer technology, as known in the art (see, for example, U.S. Pat. Nos. 6,156,338 and 6,143,729, and WO9901157 and the related devices in WO9901175).

[0104] As noted above, a number of compositions comprising one or more of the abrogen polypeptides of the invention can be prepared. Combinations of two or more isolated or purified abrogen polypeptides can be prepared. In addition, combinations of one or more abrogen polypeptides with another biologically active compound, such as a therapeutic compound, can be prepared.

[0105] The combination according to the present invention can be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above.

[0106] Therapeutic agents for possible combination are especially one or more cytostatic or cytotoxic compounds, for example a chemotherapeutic agent or several selected from the group comprising an inhibitor of polyamine biosynthesis, an inhibitor of protein kinase, especially of serine/threonine protein kinase, such as protein kinase C, or of tyrosine protein kinase, such as epidermal growth factor receptor tyrosine kinase, a cytokine, a negative growth regulator, such as TGF-β or IFN-β, an aromatase inhibitor, a classical cytostatic, and an inhibitor of the interaction of an SH2 domain with a phosphorylated protein.

[0107] The pharmaceutical compositions according to the present invention for use in a method for the prophylactic or especially therapeutic treatment of angiogenesis related disease; especially those mentioned hereinabove, as well as tumor diseases.

[0108] Preference is given to the use of solutions of the active ingredient, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilised compositions comprising the active ingredient alone or together with a carrier, for example mannitol, can be made up before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers.

[0109] Suspensions in oil comprise as the oil component the vegetable, synthetic, or semi-synthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brassidic acid or linoleic acid, if desired with the addition of antioxidants, for example vitamin E, p-carotene or 3, 5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di-or trivalent, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol. As fatty acid esters, therefore, the following are mentioned: ethyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethylene glycerol trioleate from Gattefoss, Paris), “Labrafil M 1944 CS” (unsaturated polyglycolized glycerides prepared by alcoholysis of apricot kernel oil and consisting of glycerides and polyethylene glycol ester; Gattefossé, France),“Labrasol” (saturated polyglycolized glycerides prepared by alcoholysis of TCM and consisting of glycerides and polyethylene glycol ester; Gattefossé, France), and/or “Miglyol 812” (triglyceride of saturated fatty acids of chain length C9 to C12 from Huis AG, Germany), but especially vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.

[0110] The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.

[0111] Pharmaceutical compositions for oral administration can be obtained, for example, by combining the active ingredient with one or more solid carriers, if desired granulating a resulting mixture, and processing the mixture or granules, if desired or necessary, by the inclusion of additional excipients, to form tablets or tablet cores.

[0112] Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

[0113] Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient.

[0114] Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as cornstarch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.

[0115] For parenteral administration, aqueous solutions of an active ingredient in water-soluble form, for example of a water-soluble salt, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers, are especially suitable. The active ingredient, optionally together with excipients, can also be in the form of a lyophilizate and can be made into a solution before parenteral administration by the addition of suitable solvents.

[0116] Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.

[0117] Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid.

[0118] The invention relates likewise to a process or a method for the treatment of one of the pathological conditions mentioned hereinabove, especially angiogenesis related diseases or neoplastic disease.

[0119] A polypeptide or combination can be administered as such or especially in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a patient requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.05 g to approximately 5 g, preferably from approximately 0.25 g to approximately 1.5 g, of a compound of the present invention.

EXAMPLES

[0120] Previous studies have shown that the ATF molecule can be effective as an anti-tumoral and anti-angiogenic molecule especially when delivered by gene therapy vectors [6]. However the presence of the EGF like domain may lead to the activation of intracellular pathways in both tumor cells [3] and endothelial cells [7]. These activities are counterproductive to an anti-angiogenic treatment. We have assessed the potency of the kringle domain from human and mouse uPA (ATF-kringle) by in vitro and in vivo assays for its potential as an anti-angiogenic therapeutic. The kringle domain of human uPA was previously shown to be a potent source of attraction for smooth muscle cells [2]. This activity again is counterproductive to use as an anti-angiogenic agent. Surprisingly, our data now shows that ATF-kringle containing polypeptides can inhibit endothelial cell activation and/or proliferation mediated by several different proangiogenic proteins, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and in a species independent manner. We have designated the name Abrogen to this activity.

Example 1 Cloning and Manipulating Abrogen Nucleic Acids

[0121] Exemplary primary nucleotide and polypeptide structures for both the mouse and human abrogens sequences are shown below. Amino acid sequence of human abrogen N43 ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs nal qlg SEQ ID NO.: 1 lgk hny crn pdn rrr pwc yvq vgl kpl vqe cmv hdc ad Nucleotide sequence of human abrogen N43 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ ID NO.: 2 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac agatctaatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg gtgcatgact gcgcagat Amino acid sequence of mouse abrogen ktc yhg nyd syr gka ntd tkg rpc law nap avl qkp yna hrp dai slg SEQ ID NO.: 3 lgk hny crn pdn qkr pwc yvq igl rqf vqe cmv hdc sl Nucleotide sequence of mouse abrogen aaaacctgct atcatggaaa tggtgactct taccgaggaa aggccaacac tgataccaaa SEQ ID NO.: 4 ggtcggccct gcctggcctg gaatgcgcct gctgtccttc agaaacccta caatgcccac agacctgatg ctattagcct aggcctgggg aaacacaatt actgcaggaa ccctgacaac cagaagcgac cctggtgcta tgtgcagatt ggcctaaggc agtttgtcca agaatgcatg gtgcatgact gctctctt Amino acid sequence of human abrogen ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs dal qlg SEQ ID NO.: 5 lgk hny crn pdn rrr pwc yvq vgl kpl vqe cmv hdc ad Nucleotide sequence of human abrogen D43 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ ID NO: 6 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg gtgcatgact gcgcagat Amino acid sequence of human abrogen D43 and L74 ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs dal qlg lgk SEQ ID NO: 7 hny crn pdn rrr pwc yvq vgl kll vqe cmv hdc ad Nucleotide sequence of abrogen D43 and L74 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ ID NO: 8 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc tgcttgtcca agagtgcatg gtgcatgact gcgcagat

[0122] Exemplary polypeptide sequences of the fusion proteins comprising the human abrogen having sequence of SEQ ID NO: 1 fused to the IL-2 signal peptide and to human serum albumin or immunoglobulin IgG2 Fc region, as well as linker peptide sequences, are listed below. human abrogen as secreted from pMB063 AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSDALQLGL SEQ ID NO: 9 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCAD human abrogen as secreted from pBA140 AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO: 10 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCAD human HSA amino acid sequence DAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLVNEVTEF SEQ ID NO: 11 AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP KEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGL Linker DA(G₄S)₃ DAGGGGSGGGGSGGGGS SEQ ID NO: 12 Fusion HSA—linker DA(G₄S)₃—abrogen (pMB060) ADAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLNEVTEF SEQ ID NO: 13 AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLEFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP KEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGLDAGG GGSGGGGSGG GGSKTCYEGN GHFYRGKAST DTMGRPCLPW NSATVLQQTY HAHRSNALQL GLGKHNYCRN PDNRRRPWCY VQVGLKPLVQ ECMVHDCAD Secreted fusion HSA—linker DA—abrogen from pMB059 (IL2sp is not on the sequence below but has been introduced into all the mammalian expression vector) ADAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLVNEVTEF SEQ ID NO: 14 AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP KEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGLDAKT CYEGNGHFYR GKASTDTMGR PCLPWNSATV LQQTYHAHRS NALQLGLGKH NYCRNPDNRR RPWCYVQVGL KPLVQECMVH DCAD Fusion abrogen—HSA (pMB056) AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEO ID NO: 15 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADDAH KSEVAHRFKD LGEENFKALV LIAFAQYLQQ CPFEDHVKLV NEVTEFAKTC VADESAENCD KSLHTLFGDK LCTVATLRET YGEMADCCAK QEPERNECFL QHKDDNPNLP RLVRPEVDVM CTAFHDNEET FLKKYLYEIA RRHPYFYAPE LLFFAKRYKA AFTECCQAAD KAACLLPKLD ELRDEGKASS AKQRLKCASL QKFGERAFKA WAVARLSQRF PKAEFAEVSK LVTDLTKVHT ECCHGDLLEC ADDRADLAKY ICENQDSISS KLKECCEKPL LEKSHCIAEV ENDEMPADLP SLAADFVESK DVCKNYAEAK DVFLGMFLYE YARRHPDYSV VLLLRLAKTY ETTLEKCCAA ADPHECYAKV FDEFKPLVEE PQNLIKQNCE LFEQLGEYKF QNALLVRYTK KVPQVSTPTL VEVSRNLGKV GSKCCKHPEA KRMPCAEDYL SVVLNQLCVL HEKTPVSDRV TKCCTESLVN RRPCFSALEV DETYVPKEFN AETFTFHADI CTLSEKERQI KKQTALVELV KHKPKATKEQ LKAVMDDFAA FVEKCCKADD KETCFAEEGK KLVAASQAAL GL Linker (G₄S)₃ GGGGSGGGGSGGGGS SEQ ID NO: 16 Secreted fusion abrogen—(G₄S)₃—HSA AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO: 17 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADGGG GSGGGGSGGG GSDAHKSEVA HRFKDLGEEN FKALVLIAFA QYLQQCPFED HVKLVNEVTE FAKTCVADES AENCDKSLHT LFGDKLCTVA TLRETYGEMA DCCAKQEPER NECFLQHKDD NPNLPRLVRP EVDVMCTAFH DNEETFLKKY LYEIARRHPY FYAPELLFFA KRYKAAFTEC CQAADKAACL LPKLDELRDE GKASSAKQRL KCASLQKFGE RAFKAWAVAR LSQRFPKAEF AEVSKLVTDL TKVHTECCHG DLLECADDRA DLAKYICENQ DSISSKLKEC CEKPLLEKSH CIAEVENDEM PADLPSLAAD FVESKDVCKN YAEAKDVFLG MFLYEYARRH PDYSVVLLLR LAKTYETTLE KCCAAADPHE CYAKVFDEFK PLVEEPQNLI KQNCELFEQL GEYKFQNALL VRYTKKVPQV STPTLVEVSR NLGKVGSKCC KHPEAKRMPC AEDYLSVVLN QLCVLHEKTP VSDRVTKCCT ESLVNRRPCF SALEVDETYV PKEFNAETFT FHADICTLSE KERQIKKQTA LVELVKHKPK ATKEQLKAVM DDFAAFVEKC CKADDKETCF AEEGKKLVAA SQAALGL HSA—DA(G₄S)₃—abrogen DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA SEQ ID NO: 18 KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLDAGGG GSGGGGSGGG GSKTCYEGNG HFYRGKASTD TMGRPCLPWN SATVLQQTYH AHRSNALQLG LGKHNYCRNP DNRRRPWCYV QVGLKPLVQE CMVHDCAD murine IgG2a Fe region EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED SEQ ID NO: 19 DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVN NKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYV EWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGK Fusion IgG2a-abrogen ARLEPRGPTI KPCPPCKCPA PNLLGGPSVF IFPPKIKDVL MISLSPIVTC SEQ ID NO: 20 VVVDVSEDDP DVQISWFVNN VEVHTAQTQT HREDYNSTLR VVSALPIQHQ DWMSGKEFKC KVNNKDLPAP IERTISKPKG SVRAPQVYVL PPPEEEMTKK QVTLTCMVTD FMPEDIYVEW TNNGKTELNY KNTEPVLDSD GSYFMYSKLR VEKKNWVERN SYSCSVVHEG LHNHHTTKSF SRTPGKKTCY EGNGHFYRGK ASTDTMGRPC LPWNSATVLQ QTYHAHRSNA LQLGLGKHNY CRNPDNRRRP WCYVQVGLKP LVQECMVHDC AD abrogen—RL—IgG2a AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO: 21 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADRLE PRGPTIKPCP PCKCPAPNLL GGPSVFIFPP KIKDVLMISL SPIVTCVVVD VSEDDPDVQI SWFVNNVEVH TAQTQTHRED YNSTLRVVSA LPIQHQDWMS GKEFKCKVNN KDLPAPIERT ISKPKGSVRA PQVYVLPPPE EEMTKKQVTL TCMVTDFMPE DIYVEWTNNG KTELNYKNTE PVLDSDGSYF MYSKLRVEKK NWVERNSYSC SVVHEGLHNH HTTKSFSRTP GK amino acid sequence of thioredoxin SDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY SEQ ID NO: 22 QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL KEFLDANLAG thrombin cleavage site LVPRGS SEQ ID NO: 23

[0123] The cDNA sequence can be obtained from GenBank or a number of available sources.

[0124] PCR based methods can be used to retrieve the cDNA from an appropriate library. The cDNA can then be conveniently stored in a vector such as the pGEM or pGEX vectors by standard ligation or plasmid manipulation methods. The polypeptide encoding regions are then transferred into an appropriate, selected expression cassette or vector. Specific examples of vectors for various applications exist, including gene therapy (Chen et al., Hum Gen Ther 11: 1983-96 (2000); MacDonald et al., Biochecm Biophys Res Comm 264:469-477 (1999); Cao et al., J Biol Chem 271:29461-67 (1996); Li et al., Hum Gene Ther 10:3045-53 (1999)). For the examples that follow, the method of Crouzet et al., Proc. Natl. Acad. Sci. USA 94:1414-1419 (1997), is used to prepare recombinant adenovirus with E1/E3 deletion, CMV expression promotor and SV40 polyA. The plasmid vector used below contains the Amp resistance gene, the CMV promotor, the SV40 poly A sequence, and the IL-2 signal sequence for efficient secretion. The fairly robust adenoviral system can be selected for its ability to be used in a variety of cell types, whereas the plasmid system is selected for its relative efficiency of vector introduction. One skilled in the art is familiar with selecting or modifying vectors with these or other elements for use.

[0125] Once cloned and inserted into an appropriate vector, any of the abrogen encoding sequences or abrogen derivatives encoding sequences can be assayed for specific activity related to anti-angiogenesis using the Examples below or an assay mentioned here or in the references.

[0126] In a preferred embodiment for expressing a recombinant abrogen polypeptide, a vector comprising the coding region for human serum albumin linked to the C-terminus of the abrogen encoding region is used (see, for example, Lu et al., FEBS Lett. 356: 56-9 (1994)). Other fusion proteins or chimeric proteins can also be used. In another embodiment of a fusion protein, the abrogen encoding region is linked to an immunogenic peptide or polypeptide encoding region. These fusions can be used in created antibodies or monoclonal antibodies against an abrogen. Methods for preparing antibodies are well known in the art and both the purified abrogen polypeptides and fusion of them can be used to prepare antibodies. Monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et ai., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide expressing cell. The mice splenocytes are extracted and fused with a suitable myeloma cell line, such myeloma cell line SP20, available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described (Wands et al., Gastroenterology 80:225-232 (1981)).

[0127] The hybridoma cells obtained through such a selection are then assayed to identify clones, which secrete antibodies capable of binding the polypeptide. Additional fusions can be used to ease purification of abrogen polypeptides, including poly-His tracks, constant domain of immunoglobulins (IgG), the carboxy terminus of either Myc or Flag epitope (Kodak), and glutathione-S-transferase (GST) fusions. Plasmids for this purpose are readily available.

[0128] A relatively simple method for preparing recombinant or purified abrogen polypeptide involves the baculovirus expression system or the pGEX system (Nesbit et al., Oncogene 18:6469-6476 (1999), Nesbit et al., J of Immunol 166:6483-90 (2001)). In the baculovirus system, plasmid DNA encoding the abrogen polypeptide is cotransfected with a commercially available, linearized baculovirus DNA (BaculoGold baculovirus DNA, Pharmingen, San Diego, Calif.), using the lipofection method (Felgner et al., PNAS 84:7413-7417 (1987)). BaculoGold virus DNA and the plasmid DNA are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). 10 μl Lipofectin and 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature.

[0129] The transfection mixture is added drop-wise to Sf 9 insect cells (ATCC CRL 1711), and seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The cells are cultured at 27° C. for four days. The cells can then be selected for appropriately transduction and assayed for the expression of abrogen polypeptide. If a fusion polypeptide was desired, the fusion polypeptide can be purified by known techniques and used to prepare monoclonal antibodies.

Example 2 Proliferation Analysis of Transduced HUVEC Using Alamar Blue

[0130] A number of different assays for analyzing cell proliferation, tubule formation, cell migration, endothelial cell growth, and tumor metastasis exist. Some of them are described in the references cited.

[0131] Human umbilical vein endothelial cells (HUVEC: Clonetics, San Diego) are seeded at 5×10⁵ cells/ well of 6-well-plate in EGM-2 medium. The cells are incubated overnight at 37° C., 5% CO₂. Endothelial Cell Basal Medium (EBM) and Endothelial Cell Growth Medium (EGM) are available (Clonetics, San Diego). The medium is aspirated off and 500 ul of ECM medium containing 100 IT/cell viruses put over cells. The cells are incubated at 37° C. for 2 hours, then aspirated and 1.5 ml EGM-2 medium is added. The cells are again incubate overnight at 37° C.

[0132] The cells are trypsinized, counted, and seeded at 2000cell/well of 96-well-plate in EGM-2 medium. The cells are incubated at 37° C. for 3 hours. The medium is changed into 200 μl of the following medium: Control=ECM+0.5% FBS; Test 1=control medium with bFGF 10 ng/ml; Test 2=control medium with VEGF 10 ng/ml; Test 3=control medium with bFGF 10 ng/ml+VEGF 10 ng/ml. After changing the medium, the cells are incubated at 37° C. for 5 days. 20 μl Alamar Blue (BioSource International) for each well is added. Plates are incubated at 37° C. for 6 hours and then the OD read at 570 nm and 595 nm.

[0133] Typical results are depicted in FIG. 1. From the results of this proliferation assay, both human and mouse ATF-K polypeptides (SEQ ID NO.: 1, 3, 5, and 7) are very effective in abrogating the proliferation of endothelial cells induced by bFGF and VEGF.

Example 3 Assay of Transduced HUVEC Embedded in Fibrin Gel

[0134] In an assay that distinguishes the abrogen activity from angiostatin, human umbilical vein endothelial cells (HUVEC: Clonetics, San Diego) are seeded (passage 3, growing in EGM-2 medium) at 5×10⁵ cells/ well of 6-well-plate in EGM-2 medium. The embedded cell assay also or alternatively provides data concerning the invasiveness of the endothelial cells in response to certain treatments. Endothelial cell tubule formation induced by pro-angiogenic factors such as FGF and VEGF, a characteristic measured by this assay, can be directly correlated to angiogenesis. The abrogen polypeptides used here can inhibit or reduce angiogenesis by inhibiting tubule formation. The use of virally transduced HUVEC can provide very detailed information as to the effects that a selected abrogen polypeptide or derivative has on primary cell types. The potential anti-angiogenic agents are introduced by transduction of the cells (m-ATF, h-ATF, m- ATF-K and h-ATF-K, CMV empty was included as a control) using a recombinant human adenovirus. adenovirus VP/ml vp/IT IT/ml cell/flask ul/flask Ad CMV 5.85E+12 100 5.85E+10 5.00E+06  8.55 Ad hATF 2.76E+12 100 2.76E+10 5.00E+06 18.12 Ad mATF 5.00E+12 100 5.00E+10 5.00E+06 10 Ad hATF-K 2.02E+12 161 1.25E+10 5.00E+06  5 Ad mATF-K 5.19E+12  51 1.02E+11 5.00E+06 40

[0135] The fibrin gel includes PBS (control), VEGF or bFGF. HUVEC cells are split ½ to ⅓ the day before transduction. On the day of the transduction, the cells are washed with PBS. 10 ml of serum free medium containing 100:1 (IT: cell ratio) of virus is incubated with the HUVEC for 2 hours to transduce the cells. The medium is then removed and the cells washed with PBS and 20 ml of full HUVEC medium placed in each T150 flask.

[0136] 48 hours following transduction the cells are trypsinized and the concentration of each cell solution adjusted to 5×10 ⁵ cell/ml. The assay is performed in a 24 well plate. Each well is coated with 200 μl of fibrinogen solution (12 mg/ml) and 8 ul of thrombin (50 U/ml). Then in each well is added (according to the conditions):

[0137] VEGF165 (2 μl), b-FGF(2 μl) or nothing (final [growth factor]=1 ug/ml)

[0138] Thrombin (20 ul) of a 1000 U/ml solution.

[0139] 250 μl cell solution for a final concentration of 5×₅ cells/ml

[0140] 250 μl of fibrinogen

[0141] Gels set in about 30 seconds. Then, 1.5 ml of medium is added on top. Each type of infected cells was assayed with VEGF165 alone, b-FGF alone or without any growth factor other than those already present in the medium.

[0142] After 6 days medium is removed and cells subjected to staining with Dif-Quick for enhanced visualization under microscopy. Fibrin plugs are fixed in 10% formalin, and then subjected to the 3 Dif Quick stains for 15 mins each before being rinsed in PBS and then fixed with 10% formalin again.

[0143] Representative photographs of cells are depicted in FIG. 1B. Tubules can be seen in control cells, whereas no tubules are detected in the hATF-K and mATF-K transduced cells. Tubule formation can be correlated with endothelial cell invasiveness, a characteristic of angiogenic activity. Thus, the lack of tubule formation in the abrogen polypeptide samples (human ATF Kringle and mouse ATF Kringle) demonstrates an inhibition of endothelial cell invasiveness, correlating to an inhibition of angiogenesis and metastasis. In the FIG. 1B pictures, transduced HUVEC are treated with control PBS, bFGF, or VEGF, which give the following results. For CMV control: limited structure is visible when PBS is in the fibrin gel; with VEGF there is robust proliferation showing the phenotype generated; tubules are clearly visible and are ubiquitous throughout the gel, some extensions are quite long; in the presence of bFGF the response is not as robust, the structures, which form, are long and spindle like in appearance. For full human ATF polypeptide: in PBS there are a considerable number of structures formed; the response is far more than that seen with control CMV transduced endothelial cells, also in relation to the CMV control there has been a robust response with the addition of bFGF, which is definitely synergistic with the human ATF transduced cells in comparison to those transduced with CMV; in the presence of VEGF there has been a considerable drop in the number of visible structure when compared to the CMV transduced cells. For full mouse ATF polypeptide: regardless of condition there are no structures forming in any of the gels. For human ATK Kringle (abrogen of SEQ ID NO.: 1): regardless of condition there are no structures forming in any of the gels. For mouse ATF Kringle (abrogen of SEQ ID NO.: 3): regardless of condition there are no tubule structures forming in any of the fibrin gels.

[0144] Without limiting the scope of the invention to any particular mode of action or mechanism, applicants offer the following possible explanation of these results. Human ATF still has the EGF like growth factor domain and may stimulate the growth of endothelial cells, which are human in origin. This growth is potentiated in the presence of ubiquitous bFGF in this assay, as one of the downstream effects of bFGF is the upregulation of uPAR. This synergy is observed when cells are transduced with human ATF in the presence of bFGF. In the absence of bFGF, human ATF can stimulate low level uPAR and presumably inhibits growth through the action of the kringle. Hence the observed decrease in number of structures when compared to CMV control. Mouse ATF does not cross react with human uPAR. Therefore, the mode of action is mediated through the kringle domain. With human and mouse ATF-K, there is no growth factor domain so no proliferative events can be initiated. This is specific to both bFGF and VEGF induced proliferative responses.

Example 4 In vivo Expression of Abrogen Polypeptides Using Adenoviral Vectors

[0145] For in vivo documentation of the activity of abrogen, a first experiment involves the systemic injection iv of 1×10¹¹ VP of hATF-K expressing adenovirus. Circulating levels of HATF-K as shown by Western can be measured. Exemplary expression levels at d4 can be between 500-1000 ng/ml in either SCID or SCID/Beige mice. The 4T1 spontaneously metastatic breast cell line in SCID mice is used in which animals are injected with 2×10⁵ cells sub-cutaneously in the right flank. At d7, when tumors were 20-40 mm³, adenovirus is injected at 1×10¹¹ vp: Tris, CMV1.0 control Ad; mATF-K; and hATF-K. A second and third iv administration of adenovirus can be performed. Lung metastasis is then measured at about day 35, as described below.

Example 5 In vivo Expression of Abrogen Polypeptides Using Plasmid Vectors

[0146] Two tumor models are used, employing 4T1 tumor cells and 3LL Boston tumor cells. In the assay, the anti-tumor activity of abrogen polypeptide in the prophylactic murine Lewis lung carcinoma model, 3LL-B, in C57BL/6 mice is tested. The assay is designed to assess whether circulating levels of abrogen prevent and/or reduce the formation and growth of spontaneously formed metastases from subcutaneously implanted primary tumors. The tumor cells are cultured in DMEM containing 10% FCS, sodium pyruvate, nonessential amino acids, Pen-Strep, and L-Glutamine until prepared for injection using a buffered saline solution. The tumor cells are injected into the right flank of 8-10 week old C57BL/6 or BALB/c female mice via subcutaneous injection of a suspension of 2.5×10₅ tumor cells. Six days prior to tumor cell injection, the 25 ul of the plasmid solutions (25 ug DNA in Tris EDTA with 10% glycerol) are injected into the tibialis cranialus muscle. The injection site is then exposed to 4 pulses (1 pulse per second) at 100 mV using a square wave pulse generator (the electrotransfer method, ET). Alternatively, the electrotransfer enhancement can utilize four electric pulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 msec. On about day 15 post cell injection, the primary subcutaneous tumor was surgically removed. At day 35, the lungs are collected and tumor nodules measured. Expression levels are measured on day −1, 7, and 14 relative to electrotransfer. A control alkaline phosphatase expressing plasmid (mSEAP) is used to assay expression.

[0147] The results of one set of experiments are depicted in FIGS. 4-10. The empty expression plasmid and the mSEAP control plasmid treatments resulted in many lung tumor nodules. In both the 4T1 and 3LL tumor models, the mATF-K and HATF-K abrogen polypeptides reduced the size and number of metastasis. The reduction in size and number is at least equivalent to those of the known anti-angiogenic polypeptides endostatin and angiostatin (FIG. 10).

[0148] Another set of assays with 3-LL Boston cells employing electrotransfer enhancement with four electric pulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 m sec are shown in FIG. 11. Metastases were counted using a dissecting microscope. The FIG. 11 pictures of the lungs show that the formation of spontaneous lung metastases from the primary subcutaneous tumor was significantly reduced in the two therapeutic groups receiving plasmid DNA encoding either mouse of human ATF Kringle (listed as MuPAK or HuPAK here). Lung metastases counts as well as lung weights, reflected by the diameter of the “bubble” in panel C, were reduced in both treatment groups. Delivery of plasmid DNA encoding either the murine secreted alkaline phosphatase (mSEAP) or no protein as control to the T. cranialis muscle did not result in a significant reduction of lung metastases. Similar results can be obtained in the prophylactic 4T1 mammary tumor model (data not shown).

[0149] To assess the anti-tumor activity of systemically expressed abrogen polypeptides in a human breast adenocarcinoma xenograft model of SCID/bg mice, MDA-MB-435 tumor cells are used. These cells are significantly less aggressive as compared to the 4T1 and 3LL-B syngeneic mouse tumor models. However, spontaneous lung metastases formation is established in the time frame of 35 days post subcutaneous cell injection. Subcutaneous palpable MDA-MB-435 tumors are established by injecting SCID/bg mice with 10⁶ tumor cells. On day 10 post injection, plasmid DNA was transferred to the Tibialis cranialis muscle using electrotransfer as described previously. Briefly, 25 μg of plasmid DNA (a total of 50 μg) in a 25 μl volume are injected directly into each T. cranialis muscle followed by four electric pulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 msec. The primary tumor is carefully removed when the volume reached between 250 and 350 mm3, i.e. on day 39 or 44 post cell injections depending on the growth of the primary tumor. The study is terminated on day 89 and lungs harvested carefully and fixed in Bouin's solution. Metastases are counted using a dissecting microscope. FIG. 12 shows pictures of the lungs.

[0150] Lungs from mice treated with either mouse or human AFT-Kringle containing polypeptide, FIG. 12 panels B and C, bear significantly fewer metastases compared to the control group (panel A) treated with the plasmid encoding mSEAP. Overall lung metastases counts are significantly reduced as shown in panel D. By the time of treatment at day 10, no lung metastases have been formed in the lung of SCID/bg mice, so it is most likely that the systemic expression of abrogen from the muscle prevents the formation and/or growth of distant lung metastases from the primary subcutaneous tumor. This demonstrates an inhibition of angiogenesis, a hallmark for the growth of metastatic tumors.

Example 6 Production of Derivative Abrogen Polypeptides by PCR Based Site-directed Mutagenesis

[0151] In one method for generating an abrogen derivative, four oligonucleotide primers are used. Two of these are primers that flank the ends of the cDNA (SEQ ID NO.: 2, 4, 6, or 8 ) and contain convenient restriction sites for cloning into a desired vector. The other two mutagenic primers are complementary and contain the mutation(s) of interest. Typically, the mutagenic primers overlap by about 24 base pairs. Two separate PCR reactions are performed, each using a different outside primer and a different mutagenic primer that anneal to opposite strands of the DNA template. The amplified product from both PCR reactions are purified and added to a new primerless PCR mix.

[0152] After a few PCR cycles, the two products are annealed and extended at the region of overlap yielding the derivative product. The two outside primers are then added to this mixture to amplify the cDNA product by PCR. This method can be used to introduce amino acid substitutions at any point in an abrogen sequence.

[0153] In addition to the conservative amino acid substitutions noted throughout the disclosure, one skilled in the art is familiar with numerous methods for analyzing and selecting homologs and derivative sequences to use as abrogen sequences. For example, the sequence identified as “Putative-K1 (Est)” in FIG. 2 can be identified by searching for homologs using GenBank, an EST database, or any cDNA or genomic DNA database available. The EST can be pulled from a library, PCR amplified using primers specific for the EST, or synthesized using automated methods. Once isolated, the polypeptide encoding region can be cloned into an appropriate vector and tested as described above.

Example 7 Construction of IL2sp-abrogen Polypeptide

[0154] The combined techniques of site-directed mutagenesis and PCR amplification allowed to construct a chimeric gene encoding a chimeric peptide resulting from the translational coupling between the first 20 amino acids of the interleukin 2 signal peptide, which represent a signal sequence or signal peptide that is cleaved to produce the mature factor (Tadatsugu, T. et al. (1983) Nature 302:305) and the abrogen sequences as set forth in SEQ ID NO: 4 and 6 (IL2sp-abrogen). These hybrid genes were preferably bordered in 5′ of the translational initiator ATG and in 3′ of the translational stop codon and encode chimeric proteins of the IL2sp-abrogen. The hybrid gene is cloned in the pXL2996 (FIG. 13A), under the control of the human CMV Enhancer/promoter (−522/+72) and upstream of a SV40 late poly A signal. The resulting plasmid pMB063 as described in FIG. 13B was obtained. The abrogen peptide secreted from the plasmid pMB063 retained an alanine from the IL-2 signal peptide (IL2sp) at the N-terminus, and thus contains a 87 amino acid sequence as set forth in SEQ ID NO: 9.

[0155] The hybrid nucleotide sequence comprising the interleukine 2 signal peptide sequence and the abrogen sequence as set forth in SEQ ID NO: 2 was cloned in plasmid pXL 2996 downstream of the human CMV enhancer/promoter (−522/+72) and upstream of a SV40 late poly A signal. The resulting plasmid pBA140 as described in FIG. 13C was obtained. The abrogen peptide secreted from the plasmid pBA140 also retained an alanine from the IL-2 signal peptide at the N-terminus, and thus contains a 87 amino acid sequence as set forth in SEQ ID NO: 10.

Example 8 Construction of Fusion Proteins of Abrogen and HSA

[0156] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the nucleotide sequence encoding the human HSA as set forth in SEQ ID NO: 11, a linker, and the abrogen sequence as set forth in SEQ ID NO: 2 was cloned in plasmid pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The linker DA(G₄S)₃ was used (SEQ ID NO: 12). The construct of the fusion protein IL2sp-HSA-linker-abrogen and the resulting plasmid designated pMB060 are shown in FIG. 14. The fusion protein HSA/abrogen secreted from the plasmid pMB060 has the sequence as set forth in SEQ ID NO: 13.

[0157] Another linker DA (Asp-Ala) was used. The chimeric construct of the fusion protein IL2sp-HSA-DA linker-abrogen and the resulting plasmid is designated pMB059 are displayed in FIG. 15. The fusion protein HSA/abrogen secreted from the plasmid pMB059 has the sequence as set forth in SEQ ID NO: 14.

[0158] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the abrogen nucleotide sequence as set forth in SEQ ID NO: 2, and the sequence of the human HSA (SEQ ID NO: 11), was cloned in pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB056 and construct are displayed in FIG. 16. The fusion protein abrogen- HSA secreted from the plasmid pMB056 has the sequence as set forth in SEQ ID NO: 15.

[0159] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the abrogen nucleotide sequence having the sequence as set forth in SEQ ID NO: 2, a (G₄S)₃ linker (as set forth in SEQ ID NO: 16) and the sequence of the human HSA, was cloned downstream to the human CMV promoter and upstream of a SV40 polyA. The chimeric construct of the fusion protein IL2sp-abrogen-linker-HSA and the resulting plasmid designated pMB055 are displayed in FIG. 17. The fusion protein abrogen/HSA secreted from the plasmid pMB055 has the sequence as set forth in SEQ ID NO: 17.

[0160] Alternatively, a nucleotide sequence containing from 5′ to 3′ the prepro signal of HSA, the human HSA, a sequence encoding a DA(G₄S)₃ linker and the abrogen nucleotide sequence as set forth in SEQ ID NO: 2 was cloned in the plasmid pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB060m and the fusion protein prepro HSA—human HSA- DA(G₄S)₃ linker-abrogen are displayed in FIG. 18. The fusion protein HSA/abrogen secreted from the plasmid pMB060m has the sequence as set forth in SEQ ID NO: 18.

[0161] A fusion protein encoding plasmid may also comprise the bacteriophage T7 promoter suitable for the production of the abrogen polypeptide in E coli. Such plasmids are also described in U.S. Pat. No. 6,143,518. The plasmid pYG404 as described in the Patent application EP 361 991, which comprises the sequence encoding the prepro-HSA gene, may be used. For example, the C-terminal of HSA is coupled in phase with a linker sequence and the kringle polypeptide nucleotide sequence. The resulting plasmid can also be used for production of the polypeptide in yeasts, for example.

Example 10 Construction of Fusion Proteins of Abrogen and IG2a

[0162] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the murin IgG2a Fc region (SEQ ID NO: 19) and the human abrogen nucleotide sequence having the sequence as set forth in SEQ ID NO: 2 was cloned in pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB053 and the fusion construct are displayed in FIG. 19. The fusion protein IgG2alabrogen secreted from the plasmid pMB053 has the sequence as set forth in SEQ ID NO: 20.

[0163] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the human abrogen nucleotide sequence having the sequence as set forth in SEQ ID NO: 2, the nucleotide sequence coding for a RL (Arginine-Leucine) linker, the murin (mu) IgG2a Fc region was cloned in pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB057 and the fusion construct are shown in FIG. 20. The fusion protein abrogen/IgG2a secreted from the plasmid pMB057 has the sequence as set forth in SEQ ID NO: 21.

Example 11 Construction of Plasmids Suitable for the Production of Recombinant Abrogen or Fusion Polypeptide

[0164] The plasmid pXL4128, which is represented in FIG. 21 and comprises the bacteriophage T7 promoter was also constructed, and is suitable for the production of the abrogen peptide in E coli. Such plasmids for the production in E.coli are also described in U.S. Pat. No. 6,143,518. The plasmid pYG404 as described in the Patent application EP 361 991, which comprise the sequence encoding the prepro-HSA gene may be used. For example, the C-terminal of HSA is coupled in transitional phase with a linker sequence and the abrogen nucleotide sequence. The resulting plasmid is used for production of the peptide in yeasts, for example.

Example 12 Construction of Fusion Protein Construct of trxA and Abrogen Polypeptide

[0165] The polypeptide sequence of abrogen N43 and abrogen is used for incorporation into a fusion protein. The abrogen polypeptide can then be expressed in soluble form, or substantially soluble form, in E. coli cells with the use of a bacterial expression vector, such as pET28-Trx (see FIG. 22; Novagen). K4 from angiostatin and K5 of plasminogen are also incorporated into a fusion protein and used as a control.

[0166] The sequences are amplified by PCR and the amplified fragments digested by NdeI-BamHI and cloned into pET28-Trx digested with NdeI-BamHI. Alternatively, sequences can be prepared using synthetic methods or a combination of synthetic and other methods, such as PCR or recombinant manipulation. The following Table presents the sequences selected and the primers used for cloning in an exemplary expression method. The plasmids obtained for the expression of each kringle polypeptide are also listed in the Table. Templates for the kringle sequences are available from a number of sources. Plasmid for Sequence Primers expression Abrogen N43 Sense: AAACATATGGCCAAAACCTGCTATGAGGG pXL4189 Antisense: AAAGGATCCTTAATCTGCGCAGTCATGCA Abrogen D43 Sense: AAACATATGGCCAAAACCTGCTATGAGGG pXL4215 Antisense: AAAGGATCCTTAATCTGCGCAGTCATGCA K4 from Sense: AAAAGCTTCATATGGCCCAGGACTGCTA pXL4190 angiostatin Antisense: AAATCTAGAGGATCCTTATCCTGAGCA K5 from Sense: AACATATGGAAGAAGACTGTATGTTTGGGAA pXL4219 plasminogen Antisense: CCGGATCCTTAGGCCGCA

[0167] The plasmids for expression are also described in FIGS. 23-24. These plasmids can be sequenced to verify that they encode the expected protein. Exemplary fusion proteins are represented below and comprise a TrxA sequence (from amino acid 2 to 110; see Hoog et al., Biosci. Rep. 4:917 (1984)), a poly-histidine sequence (amino acids 118 to 123), a thrombin cleavage site (amino acids 127-132), followed by the abrogen sequence or by the K4 or K5 domain. TrxA-Abrogen N43: Translation of pXL4189 1 GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDETADEY 51 QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL 101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMAKTCYE GNGHFYRGKA 151 STDTMGRPCL PWNSATVLQQ TYHAHRSNAL QLGLGKHNYC RNPDNRRRPW 201 CYVQVGLKPL VQECMVHDCA D Abrogen N43 GSHMAKTCYE GNCHFYRGKA STDTMGRPCL PWNSATVLQQ TYHAHRSNAL QLGLGKHNYC RNPDNRRRPW CYVQVGLKPL VQECMVHDCA D TrxA-Abrogen D43: Translation ofpXL4215 1 GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY 51 QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL 101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMAKTCYE GNGHFYRGKA 151 STDTMGRPCL PWNSATVLQQ TYHAHRSDAL QLGLGKHNYC RNPDNRRRPW 201 CYVQVGLKPL VQECMVHDCA D Abrogen D43 GSHM AKTCYE GNGHFYRGKA STDTMGRPCL PWNSATVLQQ TYHAHRSDAL QLGLGKHNYC RNPDNRRRPW CYVQVGLKPL VQECMVHDCA D TrxA-K4 kringle from angiostatin: Translation of pXL4190 1 GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY 51 QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL 101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMAQDCYH GDGQSYRGTS 151 STTTTGKKCQ SWSSMTPHRH QKTPENYPNA GLTMNYCRNP DADKGPWCFT 201 TDPSVRWEYC NLKKCSG K4 kringle from angiostatin GSHMAQDCYH GDGQSYRGTS STTTTGKKCQ SWSSMTPHRH QKTPENYPNA GLTMNYCRNP DADKGPWCFT TDPSVRWEYC NLKKCSG TrxA-K5 kringle from plasminogen: Translation of pXL4219 1 GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY 51 QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL 101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMEEDCMF GNGKGYRGKR 151 ATTVTGTPCQ DWAAQEPHRH SIFTPETNPR AGLEKNYCRN PDGDVGGPWC 201 YTTNPRKLYD YCDVPQCAA K5 kringle from plasminogen GSHMEEDCMF GNGKGYRGKR ATTVTGTPCQ DWAAQEPHRH SIFTPETNPR AGLEKNYCRN PDGDVGGPWC YTTNPRKLYD YCDVPQCAA

[0168] The plasmids are introduced into bacteria cells, such as E. coli BL21 λDE3trxB⁻ (Novagen). Isolated clones are inoculated in LB media containing kanamycin for selection at 37° C. After dilution, cultures are grown until an OD600 nm reaches 0.6-1.5. Expression of the fusion protein is initiated at 30° C. by adding IPTG to a final concentration of 1 mM, and continues for 3 hours. Cells are pelleted and an aliquot used to extract total protein, or to separate soluble from insoluble fractions. These samples are analyzed after separation on a polyacrylamide gel (Novex 4-12%) and staining with Coomassic Brilliant Blue.

[0169]FIG. 25 represents the results obtained with TrxA-abrogenN43 and TrxA-K4 from angiostatin. The results show that the proteins are expressed at the appropriate molecular weight (around 24 kD) and that they are soluble (around 50% for Trx-AbrogenN43 and 90% for Trx-K4). Similar results were obtained with Trx-abrogenD43 and TrxA-K5 from plasminogen.

Example 13 Purification of Abrogen From a Fusion Protein

[0170] The abrogen polypeptide can be liberated from the fusion protein using a cleavage site present in the fusion protein sequence and an appropriate cleavage enzyme. A variety of cleavage sites and related methods for cleaving a protein are available, including chemical cleavage and terminal peptidases. This example employs the thrombin cleavage site. A cell pellet of 25 grams (centrifugation pellet) from the E. coli BL21 λDE3trxB⁻ (pXL4215) cells are taken up with 100 ml of 20 mM potassium phosphate (pH 7.4)-0.5 M NaCl (buffer A), containing 12,500 units of Benzonase™, 35 mg of lysozyme, 0.1% Triton X-100 and 0.5 mM EDTA. The suspension thereby obtained is incubated for 30 min at 37° C., and then centrifuged at 12,000×g for 60 min at +4° C. The supernatant is collected and injected onto a column of Sephadex G-25 (Amersham Biosciences) equilibrated with buffer A and the protein fraction is collected and loaded onto a Hi Trap Chelating HP column (Amersham Biosciences) previously loaded with Ni²⁺ and equilibrated with buffer A containing 10 mM imidazole. The Hi Trap Chelating column is washed with buffer A containing 100 mM imidazole, and the fraction containing fusion protein is eluted with 300 mM imidazole in buffer A. This fraction is chromatographed on a Sephadex G25 column (Amersham Biosciences) equilibrated with buffer A, collected, mixed with 2 μg of thrombin per mg of protein, and incubated for 16 h at 25° C. The resulting solution is injected onto a Hi Trap Benzamidine Sepharose Fast Flow column (Amersham Biosciences), equilibrated, and eluted with buffer A. The fraction that is not retained on the column is collected and loaded onto a second Hi Trap Chelating HP column previously loaded with Ni²⁺ and equilibrated with buffer A. The liberated kringle polypeptide is eluted from the column with a linear gradient of 0 to 150 mM imidazole in buffer A over 10 column volumes. Purified kringle polypeptide is buffer exchanged by gel filtration on a column of Sephadex G25 equilibrated with PBS (pH 7.4), filtered through a 0.2 μm filter and stored at +4° C. until use.

[0171] After this step, the polypeptide is substantially purified. Gel electrophoresis analysis shows a single band by SDS-PAGE after Coomassie staining, centered at a molecular weight estimated at around 10,000. It is unambiguously identified by N-terminal sequencing (10 amino-acids). Protein concentration is quantitated by Coomassie Blue staining with the Bradford reagent. TABLE 1 Typical purification of uPA kringle from E. coli BL21 λDE3trxB⁻ (pXL4215) Volume Step (mL) Total protein (mg) Crude lysate 102 1020 First Hi Trap Chelating HP 20 113 column eluate Hi Trap benzamidine column 110 95 eluate Second Hi Trap Chelating HP 8.0 34 column eluate

[0172] The yield of purification of the peptides from the E.coli BL21 lambda DE3 trxB⁻, is set forth in the following Table 2. TABLE 2 Purification of various kringle domains from E. coli BL21 λDE3trxB⁻ Wet cell pellet Purified protein peptides Plasmid (g) obtained (mg) Abrogen (N43) pXL4189 7.5 8.7 Abrogen (D43) pXL4215 25.0 34 Angiostatin pXL4190 13.4 15.8 kringle 4 Plasminogen pXL4219 24.5 45.6 kringle 5

[0173] These data demonstrate the successful production of soluble fusion protein, in an advantageously high percentage compared to prior methods, and the successful generation of biologically active abrogen polypeptide from this fusion protein.

Example 14 Biological Activity of Kringle Domains Produced in E. coli

[0174] The biological activity of abrogen D43 kringle domain produced in E. coli was assayed on a modified three-dimensional spheroid model (see Korff et al., FASEB J. 15:447 (2001)) using 800 HUVE (Human Umbilical Vein Endothelial) cells per spheroid in a fibrin matrix in the presence of pro-angiogenic factors bFGF and VEGF. This assay measures cell tubule formation induced by pro-angiogenic factors such as FGF and VEGF, which can be directly correlated to angiogenesis. Luminized tubule formation or inhibition of tubule formation is measured 11 days after addition, to the spheroid, of pro-angiogenic factors or of pro-angiogenic factors plus the test product. The plasminogen K5 kringle domain (Cao, Y., et al., J. Biol. Chem. 272(36):22924 (1997)) and the angiostatin K4 domain (Cao Y., et al., J. Biol. Chem. 271(56): 29461(1996)), also produced in E. coli were included as positive and negative inhibitors of angiogenesis, respectively. Tubule formation in the bFGF and VEGF treated HUVE cell spheroids is markedly inhibited only in the presence abrogen D43 and Plasminogen K5 kringle domains (see FIG. 27).

REFERENCES

[0175] The references cited below may be referred to above by the reference number. Each of the references is specifically incorporate herein by reference.

[0176] 1. Andreasen, P. A., et al., The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer, 1997. 72(1): p. 1-22.

[0177] 2. Mukhina, S., et al., The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis. J Biol Chem, 2000. 275(22): p. 16450-8.

[0178] 3. Rabbani, S. A., et al., Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. J Biol Chem, 1992. 267(20): p. 14151-6.

[0179] 4. Quax, P. H., et al., Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding. Arterioscler Thromb Vasc Biol, 1998. 18(5): p. 693-701.

[0180] 5. Min, H. Y., et al., Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res, 1996. 56(10): p. 2428-33.

[0181] 6. Li, H., et al., Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice. Hum Gene Ther, 1999. 10(18): p. 3045-53.

[0182] 7. Tang, H., et al., The urokinase-type plasminogen activator receptor mediates tyrosine phosphorylation of focal adhesion proteins and activation of mitogenactivated protein kinase in cultured endothelial cells. J Biol Chem, 1998. 273(29): p. 18268-72.

[0183] 8. Soff, G. A., Angiostatin and angiostatin-related proteins. Cancer Metastasis Rev, 2000.19(1-2): p.97-107.

[0184] 9. Kleiner, D. E., Jr. and W. G. Stetler-Stevenson, Structural biochemistry and activation of matrix metalloproteases. Curr Opin Cell Biol, 1993.5(5): p.891-7.

[0185] 10. Aguirre Ghiso, J. A., et al., Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. Eur J Biochem, 1999.263(2): p.295-304.

[0186] 11. Dong, Z., et al., Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell, 1997.88(6): p.801-10.

[0187] 12. Cao, Y., et al., Kringle domains of human angiostatin. Characterization of theanti-proliferative activity on endothelial cells. J Biol Chem, 1996. 271(46): p. 29461-7.

[0188] 13. Cao, Y., et al., Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem, 1997.272(36): p.22924-8.

[0189] 14. Nesbit, M., Abrogation of tumor vasculature using gene therapy. Cancer Metastasis Rev, 2000.19(1-2): p.45-9.

[0190] 15. Lee, T. H., T. Rhim, and S. S. Kim, Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells. J Biol Chem, 1998.273(44): p.28805-12.

[0191] 16. Rhim, T. Y., et al., Human prothrombin fragment 1 and 2 inhibit bFGF-induced BCE cell growth. Biochem Biophys Res Commun, 1998.252(2): p.513-6.

[0192] 17. Xin, L., et al., Kringle 1 of human hepatocyte growth factor inhibits bovine aortic endothelial cell proliferation stimulated by basic fibroblast growth factor and causes cell apoptosis. Biochem Biophys Res Commun, 2000.277(1): p.186-90.

[0193] 18. Chen, C. T., et al., Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin. Hum Gene Ther, 2000. 11(14): p.1983-96.

[0194] The additional references below are also specifically incorporated herein by reference and cab be used, or any parts ussed, to make and use aspects of this invention.

[0195] Lee T-H, Rhim T, Kim SS. Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells. J Biol Chem 1998; 273(44): 28805-28812.

[0196] Lu H, Dhanabal M, Volk R, Waterman MJF, Ramchandran R, Knebelmann B, Segal M, Sukhatme VP. Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells. Biochem. Biophys. Res. Com. 1999; 258: 668-673.

[0197] Cao Y, Chen A, Seong Soo AA, Richard-Weidong J, Davidson D, Cao Y, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 1997; 272(36): 22924-22928.

[0198] Sauter BV, Martinet O, Zhang W-J, Mandeli J, Woo SLC. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases. Proc. Natl. Acad. Sci. 2000; 97(9): 4802-4807.

[0199] Li H, Lu H, Griscelli F, Opolon P, Sun L-Q, Ragot T, Legrand Y, Belin D, Soria J, Soria C, Perricaudet M, Yeh P. Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice. Gene Therapy 1998; 5: 1105-1113.

[0200] Dong Z, Yoneda J, Kumar R, Fidler IJ. Angiostatin-mediated suppression of cancer metastases by primary neoplasms engineered to produce granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 1998; 188(4): 755-763.

[0201] Cao Y, Ji RW, Davidson D, Schaller J, Marti D, Sohndel S, McCance SG, O'Reilly MS, Llinas M, Folkmann J. Kringle domains of human angiostatin. J. Biol. Chem. 1996; 271(56): 29461-29467.

[0202] Mukhina S, Stepanova V, Traktouev D, Poliakov A, Beabealashvilly R, Gursky Y, Minashkin M, Shevelev A, Tkachuk V. The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. J. Biol. Chem. 2000; 275(22): 16450- 16458.

[0203] Fischer K, Lutz V, Wilhelm O, Schmitt M, Graeff H, Heiss P, Nishiguchi T, Harbeck N, Luther T, Magdolen V, Reuning U. Urokinase induces proliferation of human ovarian cancer cells: characterization ofstructual elements required for growth factor function. FEBS Lett. 1998; 438(1-2): 101-105.

[0204] Koopman JL, Slomp J, de Bart AC, Quax PH, Verheijen JH. Mitogenic effects of urokinse on melanoma cells are independent of high affinity bindng to the urokinase receptor. J. Biol. Chem. 1998; 273(50): 33267-33272.

[0205] Rabbani SA, Mazar AP, Bernier SM, Haq M, Bolivar I, Henkin J, Goltzman D. Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. J. Biol. Chem. 1992; 267(20): 14151-14156.

[0206] Korff T, Kimmina S, Martiny-Baron G and Augustin H. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smopth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 2001; 15: 447-457.

1 70 1 86 PRT Artificial Sequence Human abrogen N43 1 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ala Asp 85 2 258 DNA Artificial Sequence Human abrogen N43 2 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg 60 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac 120 agatctaatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg 240 gtgcatgact gcgcagat 258 3 86 PRT Artificial Sequence Mouse abrogen 3 Lys Thr Cys Tyr His Gly Asn Gly Asp Ser Tyr Arg Gly Lys Ala Asn 1 5 10 15 Thr Asp Thr Lys Gly Arg Pro Cys Leu Ala Trp Asn Ala Pro Ala Val 20 25 30 Leu Gln Lys Pro Tyr Asn Ala His Arg Pro Asp Ala Ile Ser Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Gln Lys Arg Pro 50 55 60 Trp Cys Tyr Val Gln Ile Gly Leu Arg Gln Phe Val Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ser Leu 85 4 258 DNA Artificial Sequence Mouse abrogen 4 aaaacctgct atcatggaaa tggtgactct taccgaggaa aggccaacac tgataccaaa 60 ggtcggccct gcctggcctg gaatgcgcct gctgtccttc agaaacccta caatgcccac 120 agacctgatg ctattagcct aggcctgggg aaacacaatt actgcaggaa ccctgacaac 180 cagaagcgac cctggtgcta tgtgcagatt ggcctaaggc agtttgtcca agaatgcatg 240 gtgcatgact gctctctt 258 5 86 PRT Artificial Sequence Human abrogen 5 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ala Asp 85 6 258 DNA Artificial Sequence Human abrogen D43 6 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg 60 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac 120 agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg 240 gtgcatgact gcgcagat 258 7 86 PRT Artificial Sequence Human abrogen D43 and L74 7 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Leu Leu Val Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ala Asp 85 8 258 DNA Artificial Sequence Human abrogen D43 and L74 8 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg 60 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac 120 agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc tgcttgtcca agagtgcatg 240 gtgcatgact gcgcagat 258 9 87 PRT Artificial Sequence Human abrogen as secreted from pMB063 (abrogen D43) 9 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp 85 10 87 PRT Artificial Sequence Human abrogen as secreted from pBA140 (abrogen N43) 10 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp 85 11 585 PRT Artificial Sequence Fusion protein human abrogen 11 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 12 17 PRT Artificial Sequence Human derived linker peptide 12 Asp Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser 13 689 PRT Artificial Sequence Fusion protein human abrogen 13 Ala Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly 1 5 10 15 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 20 25 30 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 35 40 45 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 50 55 60 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 65 70 75 80 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 85 90 95 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 100 105 110 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 115 120 125 His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 130 135 140 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 145 150 155 160 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 165 170 175 Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala 180 185 190 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly 195 200 205 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 210 215 220 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 225 230 235 240 Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 245 250 255 Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 260 265 270 Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 275 280 285 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 290 295 300 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr 305 310 315 320 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 325 330 335 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 340 345 350 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 355 360 365 Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu 370 375 380 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 385 390 395 400 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 405 410 415 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 420 425 430 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 435 440 445 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 450 455 460 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 465 470 475 480 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 485 490 495 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 500 505 510 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 515 520 525 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 530 535 540 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 545 550 555 560 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 565 570 575 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Asp Ala Gly Gly Gly Gly 580 585 590 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Thr Cys Tyr Glu 595 600 605 Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly 610 615 620 Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr 625 630 635 640 His Ala His Arg Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys His Asn 645 650 655 Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln 660 665 670 Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala 675 680 685 Asp 14 674 PRT Artificial Sequence Fusion protein human abrogen 14 Ala Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly 1 5 10 15 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 20 25 30 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 35 40 45 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 50 55 60 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 65 70 75 80 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 85 90 95 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 100 105 110 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 115 120 125 His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 130 135 140 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 145 150 155 160 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 165 170 175 Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala 180 185 190 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly 195 200 205 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 210 215 220 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 225 230 235 240 Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 245 250 255 Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 260 265 270 Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 275 280 285 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 290 295 300 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr 305 310 315 320 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 325 330 335 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 340 345 350 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 355 360 365 Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu 370 375 380 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 385 390 395 400 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 405 410 415 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 420 425 430 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 435 440 445 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 450 455 460 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 465 470 475 480 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 485 490 495 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 500 505 510 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 515 520 525 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 530 535 540 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 545 550 555 560 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 565 570 575 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Asp Ala Lys Thr Cys Tyr 580 585 590 Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met 595 600 605 Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr 610 615 620 Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys His 625 630 635 640 Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val 645 650 655 Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys 660 665 670 Ala Asp 15 672 PRT Artificial Sequence Fusion protein human abrogen 15 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp Asp Ala His Lys Ser Glu Val Ala His 85 90 95 Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile 100 105 110 Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys 115 120 125 Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu 130 135 140 Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys 145 150 155 160 Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp 165 170 175 Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His 180 185 190 Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp 195 200 205 Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys 210 215 220 Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 225 230 235 240 Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys 245 250 255 Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu 260 265 270 Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala 275 280 285 Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala 290 295 300 Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys 305 310 315 320 Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp 325 330 335 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 340 345 350 Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys 355 360 365 Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu 370 375 380 Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys 385 390 395 400 Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met 405 410 415 Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu 420 425 430 Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys 435 440 445 Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 450 455 460 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu 465 470 475 480 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 485 490 495 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 500 505 510 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 515 520 525 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 530 535 540 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val 545 550 555 560 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 565 570 575 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu 580 585 590 Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg 595 600 605 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro 610 615 620 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala 625 630 635 640 Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala 645 650 655 Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 660 665 670 16 15 PRT Artificial Sequence Linker peptide 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 17 687 PRT Artificial Sequence Fusion protein human abrogen 17 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg 100 105 110 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 115 120 125 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 130 135 140 Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 145 150 155 160 Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 165 170 175 Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 180 185 190 Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 195 200 205 Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 210 215 220 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr 225 230 235 240 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 245 250 255 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln 260 265 270 Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg 275 280 285 Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 290 295 300 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 305 310 315 320 Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 325 330 335 Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 340 345 350 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 355 360 365 Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 370 375 380 Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met 385 390 395 400 Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 405 410 415 Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 420 425 430 Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 435 440 445 Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 450 455 460 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 465 470 475 480 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 485 490 495 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 500 505 510 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 515 520 525 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 530 535 540 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 545 550 555 560 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 565 570 575 Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 580 585 590 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 595 600 605 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 610 615 620 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys 625 630 635 640 Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 645 650 655 Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 660 665 670 Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 675 680 685 18 688 PRT Artificial Sequence Fusion protein human abrogen 18 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Asp Ala Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Thr Cys Tyr Glu Gly 595 600 605 Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg 610 615 620 Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His 625 630 635 640 Ala His Arg Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr 645 650 655 Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val 660 665 670 Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp 675 680 685 19 233 PRT Artificial Sequence Mouse IgG2a Fc region 19 Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro 1 5 10 15 Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 20 25 30 Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val 35 40 45 Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe 50 55 60 Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu 65 70 75 80 Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His 85 90 95 Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 100 105 110 Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser 115 120 125 Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 130 135 140 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro 145 150 155 160 Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 165 170 175 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met 180 185 190 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser 195 200 205 Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr 210 215 220 Lys Ser Phe Ser Arg Thr Pro Gly Lys 225 230 20 322 PRT Artificial Sequence Fusion protein human abrogen 20 Ala Arg Leu Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys 1 5 10 15 Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe 20 25 30 Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val 35 40 45 Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile 50 55 60 Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr 65 70 75 80 His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro 85 90 95 Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val 100 105 110 Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro 115 120 125 Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu 130 135 140 Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp 145 150 155 160 Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 165 170 175 Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser 180 185 190 Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu 195 200 205 Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His 210 215 220 His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys Lys Thr Cys Tyr 225 230 235 240 Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met 245 250 255 Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr 260 265 270 Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys His 275 280 285 Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val 290 295 300 Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys 305 310 315 320 Ala Asp 21 322 PRT Artificial Sequence Fusion protein human abrogen 21 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp Arg Leu Glu Pro Arg Gly Pro Thr Ile 85 90 95 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 115 120 125 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 130 135 140 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 145 150 155 160 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg 165 170 175 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 180 185 190 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu 195 200 205 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 210 215 220 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 225 230 235 240 Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 245 250 255 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 260 265 270 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu 275 280 285 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 290 295 300 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 305 310 315 320 Gly Lys 22 109 PRT Artificial Sequence Amino acid sequence of thioredoxin (fragment of TrxA) 22 Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val 1 5 10 15 Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys 20 25 30 Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu 35 40 45 Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro 50 55 60 Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu 65 70 75 80 Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys 85 90 95 Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly 100 105 23 6 PRT Artificial Sequence Thrombin cleavage site 23 Leu Val Pro Arg Gly Ser 1 5 24 9 PRT Artificial Sequence Purification tag 24 Ala Trp Arg His Pro Gln Phe Gly Gly 1 5 25 8 PRT Artificial Sequence Purification tag 25 Trp Ser His Pro Gln Phe Glu Lys 1 5 26 29 DNA Artificial Sequence Sense primer for Abrogen N43 26 aaacatatgg ccaaaacctg ctatgaggg 29 27 29 DNA Antisense primer for Abrogen N43 27 aaaggatcct taatctgcgc agtcatgca 29 28 29 DNA Artificial Sequence Sense primer for Abrogen D43 28 aaacatatgg ccaaaacctg ctatgaggg 29 29 29 DNA Artificial Sequence Antisense primer for Abrogen D43 29 aaaggatcct taatctgcgc agtcatgca 29 30 28 DNA Artificial Sequence Sense primer for K4 from angiostatin 30 aaaagcttca tatggcccag gactgcta 28 31 27 DNA Artificial Sequence Antisense primer for K4 from angiostatin 31 aaatctagag gatccttatc ctgagca 27 32 31 DNA Artificial Sequence Sense primer for K5 from plasminogen 32 aacatatgga agaagactgt atgtttggga a 31 33 18 DNA Artificial Sequence Antisense primer for K5 from plasminogen 33 ccggatcctt aggccgca 18 34 221 PRT Artificial Sequence TrxA-Abrogen N43 fusion protein 34 Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 115 120 125 Pro Arg Gly Ser His Met Ala Lys Thr Cys Tyr Glu Gly Asn Gly His 130 135 140 Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu 145 150 155 160 Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg 165 170 175 Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn 180 185 190 Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys 195 200 205 Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp 210 215 220 35 91 PRT Artificial Sequence Abrogen N43 35 Gly Ser His Met Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr 1 5 10 15 Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp 20 25 30 Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn 35 40 45 Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp 50 55 60 Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu 65 70 75 80 Val Gln Glu Cys Met Val His Asp Cys Ala Asp 85 90 36 221 PRT Artificial Sequence TrxA-Abrogen D43 fusion protein 36 Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 115 120 125 Pro Arg Gly Ser His Met Ala Lys Thr Cys Tyr Glu Gly Asn Gly His 130 135 140 Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu 145 150 155 160 Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg 165 170 175 Ser Asp Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn 180 185 190 Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys 195 200 205 Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp 210 215 220 37 91 PRT Artificial Sequence Abrogen D43 37 Gly Ser His Met Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr 1 5 10 15 Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp 20 25 30 Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp 35 40 45 Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp 50 55 60 Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu 65 70 75 80 Val Gln Glu Cys Met Val His Asp Cys Ala Asp 85 90 38 217 PRT Artificial Sequence TrxA-K4 kringle from angiostatin 38 Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 115 120 125 Pro Arg Gly Ser His Met Ala Gln Asp Cys Tyr His Gly Asp Gly Gln 130 135 140 Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln 145 150 155 160 Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn 165 170 175 Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala 180 185 190 Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu 195 200 205 Tyr Cys Asn Leu Lys Lys Cys Ser Gly 210 215 39 87 PRT Artificial Sequence K4 kringle from angiostatin 39 Gly Ser His Met Ala Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr 1 5 10 15 Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp 20 25 30 Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro 35 40 45 Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys 50 55 60 Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys 65 70 75 80 Asn Leu Lys Lys Cys Ser Gly 85 40 219 PRT Artificial Sequence TrxA-K5 kringle from plasminogen 40 Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 115 120 125 Pro Arg Gly Ser His Met Glu Glu Asp Cys Met Phe Gly Asn Gly Lys 130 135 140 Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln 145 150 155 160 Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu 165 170 175 Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp 180 185 190 Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu 195 200 205 Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala 210 215 41 89 PRT Artificial Sequence K5 kringle from plasminogen 41 Gly Ser His Met Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr 1 5 10 15 Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp 20 25 30 Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn 35 40 45 Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp 50 55 60 Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp 65 70 75 80 Tyr Cys Asp Val Pro Gln Cys Ala Ala 85 42 86 PRT Artificial Sequence Human kringle domain tPA-K2 42 Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His Ser 1 5 10 15 Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile Leu 20 25 30 Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys Pro 50 55 60 Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys Asp 65 70 75 80 Val Pro Ser Cys Ser Thr 85 43 86 PRT Artificial Sequence Human kringle domain tPA-K1 43 Ala Thr Cys Tyr Glu Asp Gln Gly Ile Ser Tyr Arg Gly Thr Trp Ser 1 5 10 15 Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu 20 25 30 Ala Gln Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly 35 40 45 Leu Gly Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro 50 55 60 Trp Cys Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser 65 70 75 80 Thr Pro Ala Cys Ser Glu 85 44 83 PRT Artificial Sequence Human kringle domain thrombin-K2 44 Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg Leu Ala 1 5 10 15 Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala Gln Ala 20 25 30 Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln Leu Val 35 40 45 Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val Trp Cys 50 55 60 Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu Asn Tyr 65 70 75 80 Cys Glu Glu 45 83 PRT Artificial Sequence Human kringle domain thrombin-K1 45 Gly Asn Cys Ala Glu Gly Leu Gly Thr Asn Tyr Arg Gly His Val Asn 1 5 10 15 Ile Thr Arg Ser Gly Ile Glu Cys Gln Leu Trp Arg Ser Arg Tyr Pro 20 25 30 His Lys Pro Glu Ile Asn Ser Thr Thr His Pro Gly Ala Asp Leu Gln 35 40 45 Glu Asn Phe Cys Arg Asn Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys 50 55 60 Tyr Thr Thr Asp Pro Thr Val Arg Arg Gln Glu Cys Ser Ile Pro Val 65 70 75 80 Cys Gly Gln 46 83 PRT Artificial Sequence Human kringle domain ROR2-K1 46 His Gln Cys Tyr Asn Gly Ser Gly Met Asp Tyr Arg Gly Thr Ala Ser 1 5 10 15 Thr Thr Lys Ser Gly His Gln Cys Gln Pro Trp Ala Leu Gln His Pro 20 25 30 His Ser His His Leu Ser Ser Thr Asp Phe Pro Glu Leu Gly Gly Gly 35 40 45 His Ala Tyr Cys Arg Asn Pro Gly Gly Gln Met Glu Gly Pro Trp Cys 50 55 60 Phe Thr Gln Asn Lys Asn Val Arg Met Glu Leu Cys Asp Val Pro Ser 65 70 75 80 Cys Ser Pro 47 83 PRT Artificial Sequence Human kringle domain ROR1-K1 47 His Lys Cys Tyr Asn Ser Thr Gly Val Asp Tyr Arg Gly Thr Val Ser 1 5 10 15 Val Thr Lys Ser Gly Arg Gln Cys Gln Pro Trp Asn Ser Gln Tyr Pro 20 25 30 His Thr His Thr Phe Thr Ala Leu Arg Phe Pro Glu Leu Asn Gly Gly 35 40 45 His Ser Tyr Cys Arg Asn Pro Gly Asn Gln Lys Glu Ala Pro Trp Cys 50 55 60 Phe Thr Leu Asp Glu Asn Phe Lys Ser Asp Leu Cys Asp Ile Pro Ala 65 70 75 80 Cys Asp Ser 48 81 PRT Artificial Sequence Human kringle domain Putative-K1 (Est) 48 Gly Gly Cys Phe Trp Asp Asn Gly His Leu Tyr Arg Glu Asp Gln Thr 1 5 10 15 Ser Pro Ala Pro Gly Leu Arg Cys Leu Asn Trp Leu Asp Ala Gln Ser 20 25 30 Gly Leu Ala Ser Ala Pro Val Ser Gly Ala Gly Asn His Ser Tyr Cys 35 40 45 Arg Asn Pro Asp Glu Asp Pro Arg Gly Pro Trp Cys Tyr Val Ser Gly 50 55 60 Glu Ala Gly Val Pro Glu Lys Arg Pro Cys Glu Asp Leu Arg Cys Pro 65 70 75 80 Glu 49 84 PRT Artificial Sequence Human kringle domain plasminogen-K5 49 Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala 1 5 10 15 Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro 20 25 30 His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu 35 40 45 Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp 50 55 60 Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro 65 70 75 80 Gln Cys Ala Ala 50 77 PRT Artificial Sequence Human kringle domain Neurotrypsin-K1 50 Trp Gly Cys Pro Ala Gly Glu Pro Trp Val Ser Val Thr Asp Phe Gly 1 5 10 15 Ala Pro Cys Leu Arg Trp Ala Glu Val Pro Pro Phe Leu Glu Arg Ser 20 25 30 Pro Pro Ala Ser Trp Ala Gln Leu Arg Gly Gln Arg His Asn Phe Cys 35 40 45 Arg Ser Pro Asp Gly Ala Gly Arg Pro Trp Cys Phe Tyr Gly Asp Ala 50 55 60 Arg Gly Lys Val Asp Trp Gly Tyr Cys Asp Cys Arg His 65 70 75 51 83 PRT Artificial Sequence Human kringle domain MSP-K4 51 Gln Asp Cys Tyr His Gly Ala Gly Glu Gln Tyr Arg Gly Thr Val Ser 1 5 10 15 Lys Thr Arg Lys Gly Val Gln Cys Gln Arg Trp Ser Ala Glu Thr Pro 20 25 30 His Lys Pro Gln Phe Thr Phe Thr Ser Glu Pro His Ala Gln Leu Glu 35 40 45 Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Ser His Gly Pro Trp Cys 50 55 60 Tyr Thr Met Asp Pro Arg Thr Pro Phe Asp Tyr Cys Ala Leu Arg Arg 65 70 75 80 Cys Ala Asp 52 83 PRT Artificial Sequence Human kringle domain MSP-K3 52 Val Ser Cys Phe Arg Gly Lys Gly Glu Gly Tyr Arg Gly Thr Ala Asn 1 5 10 15 Thr Thr Thr Ala Gly Val Pro Cys Gln Arg Trp Asp Ala Gln Ile Pro 20 25 30 His Gln His Arg Phe Thr Pro Glu Lys Tyr Ala Cys Lys Asp Leu Arg 35 40 45 Glu Asn Phe Cys Arg Asn Pro Asp Gly Ser Glu Ala Pro Trp Cys Phe 50 55 60 Thr Leu Arg Pro Gly Met Arg Ala Ala Phe Cys Tyr Gln Ile Arg Arg 65 70 75 80 Cys Thr Asp 53 82 PRT Artificial Sequence Human kringle domain MSP-K2 53 Ala Ala Cys Val Trp Cys Asn Gly Glu Glu Tyr Arg Gly Ala Val Asp 1 5 10 15 Arg Thr Glu Ser Gly Arg Glu Cys Gln Arg Trp Asp Leu Gln His Pro 20 25 30 His Gln His Pro Phe Glu Pro Gly Lys Phe Leu Asp Gln Gly Leu Asp 35 40 45 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Arg Pro Trp Cys Tyr 50 55 60 Thr Thr Asp Pro Gln Ile Glu Arg Glu Phe Cys Asp Leu Pro Arg Cys 65 70 75 80 Gly Ser 54 81 PRT Artificial Sequence Human kringle domain MSP-K1 54 Arg Thr Cys Ile Met Asn Asn Gly Val Gly Tyr Arg Gly Thr Met Ala 1 5 10 15 Thr Thr Val Gly Gly Leu Pro Cys Gln Ala Trp Ser His Lys Phe Pro 20 25 30 Asn Asp His Lys Tyr Thr Pro Thr Leu Arg Asn Gly Leu Glu Glu Asn 35 40 45 Phe Cys Arg Asn Pro Asp Gly Asp Pro Gly Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Ala Val Arg Phe Gln Ser Cys Gly Ile Lys Ser Cys Arg 65 70 75 80 Glu 55 87 PRT Artificial Sequence Human kringle domain Hyaluronan BP-K1 55 Asp Asp Cys Tyr Val Gly Asp Gly Tyr Ser Tyr Arg Gly Lys Met Asn 1 5 10 15 Arg Thr Val Asn Gln His Ala Cys Leu Tyr Trp Asn Ser His Leu Leu 20 25 30 Leu Gln Glu Asn Tyr Asn Met Phe Met Glu Asp Ala Glu Thr His Gly 35 40 45 Ile Gly Glu His Asn Phe Cys Arg Asn Pro Asp Ala Asp Glu Lys Pro 50 55 60 Trp Cys Phe Ile Lys Val Thr Asn Asp Lys Val Lys Trp Glu Tyr Cys 65 70 75 80 Asp Val Ser Ala Cys Ser Ala 85 56 83 PRT Artificial Sequence Human kringle domain HGF-K4 56 Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser 1 5 10 15 Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu 20 25 30 Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn 35 40 45 Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys 50 55 60 Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg 65 70 75 80 Cys Glu Gly 57 83 PRT Artificial Sequence Human kringle domain HGF-K3 57 Thr Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn 1 5 10 15 Thr Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro 20 25 30 His Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg 35 40 45 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe 50 55 60 Thr Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn 65 70 75 80 Cys Asp Met 58 82 PRT Artificial Sequence Human kringle domain HGF-K2 58 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 1 5 10 15 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 20 25 30 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 35 40 45 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 50 55 60 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 65 70 75 80 Ala Asp 59 83 PRT Artificial Sequence Human kringle domain HGF-K1 59 Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser 1 5 10 15 Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro 20 25 30 His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln 35 40 45 Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys 50 55 60 Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln 65 70 75 80 Cys Ser Glu 60 86 PRT Artificial Sequence Human kringle domain HGF activator-K1 60 Glu Arg Cys Phe Leu Gly Asn Gly Thr Gly Tyr Arg Gly Val Ala Ser 1 5 10 15 Thr Ser Ala Ser Gly Leu Ser Cys Leu Ala Trp Asn Ser Asp Leu Leu 20 25 30 Tyr Gln Glu Leu His Val Asp Ser Val Gly Ala Ala Ala Leu Leu Gly 35 40 45 Leu Gly Pro His Ala Tyr Cys Arg Asn Pro Asp Asn Asp Glu Arg Pro 50 55 60 Trp Cys Tyr Val Val Lys Asp Ser Ala Leu Ser Trp Glu Tyr Cys Arg 65 70 75 80 Leu Glu Ala Cys Glu Ser 85 61 83 PRT Artificial Sequence Human kringle domain Facto XII-K1 61 Ala Ser Cys Tyr Asp Gly Arg Gly Leu Ser Tyr Arg Gly Leu Ala Arg 1 5 10 15 Thr Thr Leu Ser Gly Ala Pro Cys Gln Pro Trp Ala Ser Glu Ala Thr 20 25 30 Tyr Arg Asn Val Thr Ala Glu Gln Ala Arg Asn Trp Gly Leu Gly Gly 35 40 45 His Ala Phe Cys Arg Asn Pro Asp Asn Asp Ile Arg Pro Trp Cys Phe 50 55 60 Val Leu Asn Arg Asp Arg Leu Ser Trp Glu Tyr Cys Asp Leu Ala Gln 65 70 75 80 Cys Gln Thr 62 86 PRT Artificial Sequence Human kringle domain ATF-Kringle (Abrogen) 62 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ala Asp 85 63 82 PRT Artificial Sequence Human kringle domain ApoArgC-K1 63 Gln Glu Cys Tyr His Ser Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Phe 1 5 10 15 Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 20 25 30 His Gln His Ser Arg Thr Pro Glu Lys Tyr Pro Asn Asp Gly Leu Ile 35 40 45 Ser Asn Tyr Cys Arg Asn Pro Asp Cys Ser Ala Gly Pro Trp Cys Tyr 50 55 60 Thr Thr Asp Pro Asn Val Arg Trp Glu Tyr Cys Asn Leu Thr Arg Cys 65 70 75 80 Ser Asp 64 82 PRT Artificial Sequence Human kringle domain Angiostatin-K4 64 Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser 1 5 10 15 Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro 20 25 30 His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr 35 40 45 Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe 50 55 60 Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys 65 70 75 80 Ser Gly 65 82 PRT Artificial Sequence Human kringle domain Angiostatin-K3 65 Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala 1 5 10 15 Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro 20 25 30 His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp 35 40 45 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His 50 55 60 Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 65 70 75 80 Asp Ser 66 82 PRT Artificial Sequence Human kringle domain Angiostatin-K2 66 Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser 1 5 10 15 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro 20 25 30 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 35 40 45 Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe 50 55 60 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 65 70 75 80 Thr Thr 67 83 PRT Artificial Sequence Human kringle domain Angiostatin-K1 67 Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser 1 5 10 15 Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro 20 25 30 His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu 35 40 45 Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys 50 55 60 Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu 65 70 75 80 Cys Glu Glu 68 9 PRT Artificial Sequence Human kringle domain Consensus corresponding to position 54-62 68 Asn Tyr Cys Arg Asn Pro Asp Gly Asp 1 5 69 6 PRT Artificial Sequence Human kringle domain Consensus corresponding to position 65-70 69 Gly Pro Trp Cys Tyr Thr 1 5 70 6 PRT Artificial Sequence Human kringle domain Consensus corresponding to position 77-82 70 Val Arg Trp Glu Tyr Cys 1 5 

What is claimed is:
 1. A method of inhibiting angiogenesis in a subject comprising administering an abrogen polypeptide or a fusion construct thereof to a subject.
 2. The method of claim 1, wherein the amino acid sequence of the abrogen polypeptide consists of one of SEQ ID NO.: 1, 3, 5, 7, 9, or 10 or the fusion construct consists of one of SEQ ID NO: 13, 14, 15, 17, 18, 20, or
 21. 3. A method of expressing a soluble abrogen polypeptide-containing fusion protein comprising providing a vector or nucleic acid encoding a fusion protein, which comprises a thioredoxin sequence and an abrogen polypeptide sequence, whereby the fusion protein can be expressed in a bacterial cell, inserting the vector or nucleic acid into a bacterial cell to express the fusion polypeptide, and detecting the presence of soluble fusion protein.
 4. The method of claim 3, wherein a substantial fraction of the total fusion protein is expressed in a soluble, stable form.
 5. The method of claim 4, wherein the bacterial cell is E. coli, the thioredoxin has the sequence of SEQ ID NO: 22, and the abrogen polypeptide has the sequence of one of SEQ ID NO: 1, 3, 5, 7, 9, or
 10. 6. A method of preparing an abrogen polypeptide composition, comprising the method of claim 5, wherein the vector or nucleic acid further comprises a proteolytic cleavage site for liberating the abrogen polypeptide sequence from the fusion polypeptide, and further comprising incubating the fusion protein with an appropriate cleavage enzyme to generate abrogen polypeptide molecules.
 7. The method of claim 6, wherein the cleavage site is a thrombin cleavage site.
 8. The method of claim 6, wherein the nucleic acid further comprises a purification tag placed in between the thioredoxine sequence and the cleavage site, and further comprising purifying the abrogen polypeptide from other components by chromatography.
 9. The method of claim 8, further comprising adding a pharmaceutically acceptable excipient or carrier to the purified abrogen polypeptide.
 10. The abrogen polypeptide obtained by the method of claim 8, wherein the abrogen peptide is substantially soluble and stable.
 11. A composition comprising the abrogen polypeptide of claim 10, and a suitable carrier or excipient.
 12. A method of inhibiting angiogenesis comprising administering an effective an animal or cell. 